Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof

ABSTRACT

The invention relates to neuromodulatory oligomers of the amyloid-β(1-42) protein, a particular production method, by means of which the oligomer can be obtained in a reproducible manner at high yield, the use of the oligomers as diagnostic and therapeutics agents, for the generation of oligomer-specific antibodies and for the discovery of substances which can interact with the oligomers and in the formation thereof. Corresponding methods for the production of the antibodies and for discovery of the substances are also disclosed as are the antibodies themselves and the use of the antibodies or substances as diagnostic and therapeutic agents. The invention further relates to derivatives of the oligomers and oligomers based on abbreviated forms of the amyloid-β(1-42) proteins, the production and use thereof.

The invention relates to neuromodulating oligomers of the amyloidβ(1-42) protein, to a specific method of preparation by which saidoligomers are obtainable in a reproducible manner and with high yield,and to the use of said oligomers as diagnostic and therapeutic agents,for generating oligomer-specific antibodies and for finding substancescapable of interacting with said oligomers and with the formationthereof.

The invention likewise also relates to derivatives of said oligomers, inparticular to crosslinked oligomers and oligomers based on truncatedforms of the amyloid β(1-42) protein thereof, to the preparation thereofand the use thereof.

Corresponding methods of generating the antibodies and of finding thesubstances are also described as are the antibodies themselves and theuse of said antibodies and substances as diagnostic and therapeuticagents.

Amyloid β(1-42) protein, also referred to as Aβ(1-42) for short, is acentral component of insoluble extracellular depositions (senile orneuritic plaques) composed of proteins, lipids, carbohydrates and saltsin the brains of Alzheimer and Down's syndrome patients (C. L. Masterset al. PNAS 82, 4245 - 4249, 1985). This protein which tends topolymerize in an aqueous environment may be present in very differentmolecular forms.

A simple correlation of the deposition of insoluble protein with theoccurrence or progression of dementia disorders such as, for example,Alzheimer's disease has proved to be unconvincing (R. D. Terry et alAnn. Neurol. 30. 572-580 (1991), D. W. Dickson et al. Neurobiol. Aging16, 285-298 (1995)). In contrast, the loss of synapses and cognitiveperception seems to correlate better with soluble forms of Aβ(1-42) (L.F. Lue et al. Am. J. Pathol. 155, 853-862,(1999), C. A. McLean et al.Ann. Neurol. 46, 860-866 (1999)).

With the soluble forms of Aβ(1-42), there are essentially two differenthypotheses regarding the molecular forms supposedly to be the cause fordementia disorders such as Alzheimer's disease.

Firstly, a cytotoxic action of Aβ(1-42) protofibrils is postulated. Thelatter are still soluble, fibrillar relatively highly aggregatedAβ(1-42) forms having molecular weights in the range from 150-250 kDa(Arispe et al. PNAS 90. 567 (1993), Lashuel et al., Nature 418, 291(2002)) which, due to pore-forming properties, apparently cause anuncontrolled calcium influx through the membranes of neuronal cells.

Secondly, oligomeric Aβ(1-42) derivatives having molecular weights inthe range from 15-30 kDa are described (M. P. Lambert et al. PNAS 95,6448-6453 (1998)). These nonfibrillar oligomers also referred to asamyloid derived, diffusible and dementing ligands (ADDL's for AmyloidDerived Dementing Ligands, cf. U.S. Pat No. 6,218,506 and WO 01/10900,or for Amyloid Derived Diffusible Ligands, cf. Lambert et al. supra) canbe found in preparations showing an inhibiting influence on the rate oflong-term potentiation of neurons in hippocampal sections.

However, the state of previous research on oligomers is characterized bygreat uncertainty over the actually relevant species. The information inthe literature differs greatly. Thus, U.S. Pat No. 6,218,506 describesADDLs having from 3 to 12 subunits, whereas the ADDLs described in WO01/10900 may have up to 24 subunits.

More specifically, at least two forms are discussed which have in agel-electrophoretic analysis under nondenaturing conditions molecularweights in the range from 27 to 28 kDa and 23 to 24 kDa (U.S. Pat. No.6,218,506) or from about 26 kDa to 28 kDa (WO 01/10900) and,respectively, 17 kDa and 27 kDa (M. P. Lambert et al. PNAS 95, 6448-6453(1998)) and in an SDS gel-electrophoretic analysis under denaturingconditions molecular weights of 17 kDa and 22 kDa (M. P. Lambert et al.PNAS 95, 6448-6453 (1998)) and, respectively, of from about 22 kDa toabout 24 kDa and from about 18 kDa to about 19 kDa (WO 01/10900).SDS-stable Aβ(1-42) oligomers having molecular weights in the range from8 kDa and 12 kDa have also been detected previously by Western blotmethods on brains of Alzheimer patients (C. A. McLean et al. Ann.Neurol. 46, 860-866 (1999)).

The preparation protocols described have the disadvantage of resultingin inhomogeneous oligomer preparations.

Thus it has not been possible yet to provide in a reproducible manner,let alone unambiguously identify, the molecular forms of Aβ(1-42)responsible for neuromodulation.

However, the importance of a homogeneous preparation can be gauged, forexample, from the course of a first clinical study of activeimmunization on Alzheimer patients. Vaccination with a pre-aggregatedform of Aβ(1-42) resulted in considerable side effects(mengioencephalitis, hemorrhages) in some of the patients, since theantibodies formed also recognized the Aβ(1-42) forms presumably requiredfor cell lining, resulting in inflammatory reactions (D. Schenk.; Nat.Rev. Neurosci. 3, 824-828 (2002)).

A custom therapy which especially aims at neutralizing the actuallydamaging protein form therefore absolutely requires the identificationand defined preparation of the latter.

In addition, the occurrence of N-terminally truncated forms of theAβ(1-42) protein in connection with Alzheimer's disease has beenreported. Apart from Aβ(1-42) N-terminally truncated forms were alsodetected in the depositions of brains of deceased Alzheimer patients asearly as 1985 (C. Masters et al., PNAS 82, 4245-4249 (1985)). Thus,particular proteases present in the brain, such as neprilysin (NEP24.11) or IDE (short for insulin degrading enzyme), are also known to beable to degrade Aβ(1-42) (D. J. Selkoe, Neuron 32, 177-180, (2001)).

However, the importance of the N-terminally truncated forms in thepathogenesis of Alzheimer's disease is unclear (E. B. Lee et al, JBS278, 4458-4466 (2003)). Interestingly, some patients suffering fromsporadic or familial Alzheimer's disease or Down's syndrome preferablyaccumulate these truncated forms (J. Naslund et al, PNAS 91, 8378-8382,(1994), C. Russo et al., Nature 405, 531-532, (2000), T. C. Saido et al,Neuron 14, 457-466, (1995)). A relatively recent study (N. Sergeant etal, J. of Neurochemistry 85, 1581-1591, (2003)) showed that 60% of allinsoluble Aβ peptides in the brains of deceased Alzheimer patients arebased on N-terminally truncated forms.

Antibodies directed against monomeric Aβ(1-42) protein and particularfragments thereof have already been described.

Thus, WO 03/016466 and WO 03/016467 relate to the monoclonal antibody266 and analogs thereof which lack a particular glycosylation site inCDR2. Humanized versions thereof (Hu-266) are also known. Thesedocuments also mention other monoclonal antibodies which, like antibody226, recognize epitopes in the region of amino acids 13-28 of Aβ(1-42).These include the antibodies 4G8 (also mentioned in Lue et al. (1999),supra) and 1C2. Furthermore, McLean et al. (1999), supra, mentions themonoclonal antibody 1 E8 which apparently recognizes an epitope in theregion of amino acids 18-22 of Aβ(1-42).

Moreover, a number of further antibodies are known which recognizeepitopes of N-terminal sequences of Aβ(1-42). These include commerciallyavailable, monoclonal antibody 6E10 (also mentioned in WO 01/10900;Naslund et al. (1994), supra; Sergeant et al. (2003), supra) and themonoclonal antibodies 3D6 and 10D5 (see WO 03/016467) and also Ban50andNAB228 (Lee et al. (2003), supra).

Furthermore, mention must be made of the monoclonal antibodies W02,21F12 and 3D6 and of the polyclonal serum ADA42 (Sergeant et al. (2003),supra).

An overview over anti-Aβ(1-42) antibodies currently undergoingpreclinical testing can be found in Schenk et al. (2002), supra.

The present invention is based on the object of providing the molecularforms which cause the neuromodulating and, in particular,neuron-damaging action of Aβ(1-42) and whose presence is increased indementia disorders such as Alzheimer's disease and Down's syndrome andachieves said object by means of particular Aβ(1-42) oligomers which areobtainable in the form of homogeneous preparations from monomericAβ(1-42) protein, using a special method, and by means of particularderivatives of said oligomers, in particular crosslinked oligomers andoligomers based on truncated Aβ(1-42) forms. The method of preparationenables peptide-synthetic Aβ(1-42) protein which is poorly soluble inaqueous media to be converted to defined soluble oligomers with highyield.

The present invention therefore relates to the subject matters definedin the claims.

Thus, the present invention relates to oligomers of the amyloid β(1-42)protein, said oligomers having an apparent molecular weight of about 15kDa, 20 kDa, 38 kDa or 48 kDa in SDS gel electrophoresis, or toderivatives of said oligomers having a molecular weight which may or maynot have changed according to the derivatization.

Amyloid β(1-42) protein is a polypeptide having 42 amino acids which isderived from the amyloid precursor protein (APP) by proteolyticprocessing. This also includes, in addition to human variants, isoformsof the amyloid β(1-42) protein present in organisms other than humans,in particular other mammals, especially rats.

According to a particular embodiment, the present invention relates tooligomers of human amyloid β(1-42) proteins. Human amyloid β(1-42)proteins include in particular the protein having the amino acidsequence SEQ ID NO:1 and also muteins and allelic variants thereofderived from said sequence in particular by amino acid exchange. In thisconnection, very particular mention must be made of the following aminoacid substitutions: A21G, E22K, E22Q, E22G and D23N. Thus, the muteinsor allelic variants of the amyloid β(1-42) protein include according tothe invention in particular proteins having an amino acid sequence SEQID NO:1 in which one or more amino acids selected from among alanine 21,glutamic acid 22 and aspartic acid 23 have been substituted by adifferent amino acid preferably selected from among glycine, lysine,glutamine and asparagine. Particularly important according to theinvention are substitutions in position 22, in particular by glutamineor glycine.

According to another particular embodiment, the present inventionrelates to oligomers of rat amyloid β(1-42) proteins. Rat amyloidβ(1-42) proteins include in particular the protein having the amino acidsequence SEQ ID NO:2 and also muteins and allelic variants thereofderived from said sequence in particular by amino acid exchange. In thisconnection, very particular mention must be made of those amino acidsubstitutions which correspond to the amino acid substitutions discussedfor the human sequence.

Amyloid β(1-42) protein may be prepared by known peptide-syntheticmethods or recombinantly. In addition, a number of these proteins arecommercially available. The same applies also to muteins and allelicvariants.

The oligomers of the invention are obtainable by oligomerization ofamyloid β(1-42) protein. The oligomerization comprises a noncovalentaggregation of monomeric amyloid protein so that the oligomers of theinvention can be assumed to be composed of a plurality of amyloidβ(1-42) protein monomers.

Depending on the degree of oligomerization, the oligomers of theinvention have different molecular weights. Thus it is possible toassign apparent molecular weights to said oligomers by means ofdenaturing gel electrophoresis. Said molecular weights are about 15 kDafor the oligomer A1, about 20 kDa for the oligomer A2, about 38 kDa forthe oligomer B1 and about 48 kDa for the oligomer B2, when gelelectrophoresis is carried out under standard denaturing conditions(Tris-glycine gel, 4-20%, cf. Lämmli UK, Nature 227, 680-685 (1970)),with the following standard proteins having the following apparentmolecular weights under identical conditions: myosin 250 kDa, bovineserum albumin 98 kDa, glutamine hydrogenase 64 kDa, carboanhydrase 36kDa, myoglobin 30 kDa, lysozyme 16 kDa, aprotinin 6 kDa, insulin B chain4 kDa (cf. Blue Pre-stained Standard). According to another aspect, themolecular weights for the oligomers B are from about 64 to 90 kDa, whengel electrophoresis is carried out under standard native conditions(Tris glycine gel, 4-20%), with the following standard proteins havingthe following apparent molecular weights under identical conditions:myosin 250 kDa, bovine serum albumin 98 kDa, glutamine hydrogenase 64kDa, carboanhydrase 36 kDa, myoglobin 30 kDa, lysozyme 16 kDa, aprotinin6 kDa, insulin B chain 4 kDa (cf. Blue Pre-stained Standard).

According to another aspect, the oligomers of the invention arecharacterized by an affinity for neuronal cells. It can be assumed thatsaid oligomers bind to particular cell surface proteins, in particularreceptors.

Accordingly, the present invention also relates to a method ofdetermining the binding of an oligomer of the invention to a predefinedcellular structure, which method comprises

-   -   i) allowing said oligomer of the invention to act on said        cellular structure and    -   ii) determining whether said oligomer of the invention binds to        said cellular structure.

According to another aspect, the oligomers of the invention arecharacterized by a neuromodulating action. This neuromodulating actioncan manifest itself in particular in a reduced survivability of neuronalcells, for example neuroblastoma cells (neuro-toxicity) when at leastone oligomer of the invention is allowed to act on a culture of thesecells. It is possible here to assess the survivability of said cells ina manner known per se, for example by determining the extent ofapoptosis caused by the action of the oligomers of the invention. Forthis purpose, suitable assay methods, for example colorimetric methodsbased on 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) are available. This neuromodulating action can manifest itself invivo in particular in a modulation of the firing rate of neurons.

Accordingly, the present invention also relates to a method ofdetermining the activity, in particular neurotoxicity, of an oligomer ofthe invention, which method comprises

-   -   i) allowing said oligomer of the invention to act on cells and    -   ii) determining whether the state of said cells is modified.

It is possible to provide for said methods oligomer of the invention inthe above-described manner, for example in the form of any of theabovementioned compositions. The cells or cellular structures areconveniently provided in vitro, in particular as cell culture or else,in cellular structures, as homogenates. Here, neuronal cells and, inparticular, neuroblastoma cells serve to determine neuro-toxicity.Alternatively, it is also possible to provide the cells in vivo, inparticular as part of an organism, for example of an experimentalanimal, or ex vivo.

The state of the cells is usually determined at least once prior to andat least once after the action of the oligomer. If comparison betweenthe state prior to and the state after the action results in adeviation, the oligomer tested has activity.

The type of the state to be determined depends on the type of theactivity to be determined. Neurotoxicity can be determined bydetermining the survivability, for example. For this purpose, theproportion of living cells based on the total number of cells prior toand after the action of the oligomer can be determined and compared toone another.

Based upon the methods above of determining binding or activity, it ispossible, according to particular embodiments, to test substances onwhether they inhibit binding of oligomers of the invention and/ormodulate, i.e. in particular reduce or essentially completely inhibit,or enhance the activity thereof.

For this purpose, the method is in principle carried out at least twice,once in the presence of the test substance and once more without testsubstance. To this end, said test substance is added to the oligomersusually after they have been provided.

However, if it is intended to determine whether or not a substance to betested influences formation of said oligomers, said substance isexpediently added to the reactant(s) used for oligomer formation,already prior to formation of said oligomers, i.e. already before theyhave been provided. Thus it is possible to carry out the preparationmethod of the invention with addition of the test substance and then todetermine whether and to what extent the oligomers are formed. For thispurpose, it can be determined whether the method products obtained inthis way have the properties of the oligomers of the invention, i.e.their molecular weight, binding ability and activity, for example.

For the purposes of a neuromodulating action as pronounced as possible,preference must be given to the oligomers B of the invention. In thisregard, preference is also given to derivatives of these oligomers, withparticular emphasis being given to the oligomers based on anN-terminally truncated Aβ(1-42) protein.

For process engineering reasons, the oligomers A and the oligomers B maybe produced as a mixture in the form of compositions which, in additionto the oligomers, further comprise small proportions of otherpolypeptides, in particular monomeric amyloid β(1 -42) protein and,where appropriate, also higher molecular weight forms of aggregatedamyloid β(1-42) protein. Compositions of this kind are likewise subjectmatter of the present invention and are distinguished in particular bythe proportion of oligomer(s) of the invention being at least 70% byweight, preferably at least 90% by weight, and in particular at least95% by weight, based on the totality of proteins derived from amyloidβ(1-42) protein. In a preparation of oligomers A, the proportion ofoligomer A2 (20 kDa band in the SDS gel) is at least 50% by weight andpreferably at least 70% by weight and in particular at least 85% byweight. In a preparation of oligomers B, the proportion of oligomers B1and B2 (38 kDa and 48 kDa bands in the SDS gel) is at least 60% byweight, preferably at least 75% by weight and in particular at least 90%by weight.

The oligomers of the invention may also be derivatized. The purpose ofsuch derivatizations may be, for example, to modulate thephysicochemical properties of said oligomers, in particular with respectto bioavailability, to provide said oligomers with a detectable label orto immobilize said oligomers, for example to be able to couple them tosupports. Labeling and immobilization are particularly important fordiagnostic applications.

Suitable labels familiar in the protein-biochemical field aresufficiently known to the skilled worker. These include fluorescentlabels, for example particular fluorescein and tetramethylrhodaminederivatives, luminescent labels, colorimetric labels, radio labels andmagnetic labels and also labels with affinity for complementary bindingpartners, such as biotin and streptavidin derivatives.

With respect to the apparent molecular weights, it must be taken intoaccount that the oligomer derivatives have molecular weights which mayhave increased correspondingly, compared to the nonderivatizedoligomers, but with the aggregation number being identical. Thus, forexample, a biotin derivative based on the Aβ(1-42) oligomer B1 having amolecular weight of 38 kDa in the SDS gel has a molecular weight of 42kDa.

According to a particular embodiment of the present invention, theoligomers are crosslinked. Suitable crosslinkers are known to theskilled worker and are usually bifunctional reagents such asformaldehyde, glutardialdehyde, disuccinimidyl suberate,dithiobis(succinimidyl propionate), disuccinimidyl tartrate,disulfo-succinimidyl tartrate, dimethyl adipimidate, dimethylpimelidate, dimethyl suberimidate, dimethyl3,3′-dithiobispropionimidate, N-y-maleinimidobutyloxy-succinimide ester,succinimidyl 4(N-maleinimidomethyl)cyclohexane-1-carboxylate,N-succinimidyl (4-iodacetyl)aminobenzoate and N-succinimidyl3-(2-pyridyldithio)-propionate.

Such crosslinked oligomers have the advantage that they are stabilizedand that their oligomerization is usually no longer reversible. They aretherefore particularly suitable for use in diagnostic test systems or asimmunogen for production of oligomer-specific antibodies.

The derivatives of oligomers of the invention also include oligomers offragments of the amyloid β(1-42) protein. Preference is given to thosefragments which are obtainable by the action of naturally occurringproteases. The fragments obtainable by proteolysis in physiologicalbuffers under nondenaturing conditions, in particular, have increasedproteolytic stability compared to the amyloid β(1-42) protein.Preference is given to fragments obtainable by the action ofendopeptidases. According to a particular embodiment of the presentinvention, said fragments are obtainable by the action of trypsin,chymotrypsin, thermolysin, elastase, papain or endoproteinase GluC.According to another aspect, preference is given to those fragmentswhose oligomers of the invention are distinguished by a neuromodulatingaction. For further illustration of this aspect, reference is made tothe corresponding comments on the neuromodulating action of inventiveoligomers of the amyloid β(1-42) protein.

Structurally, preferred fragments are in particular characterized inthat they are derived from the amyloid β(1-42) protein by removal ofN-terminal sequences. According to one aspect, said N-terminal sequencesmay be fragments having up to 23, preferably having up to 21 and inparticular having up to 19, amino acids of the N-terminal sequence ofthe amyloid β(1-42) protein. Accordingly, preference is given accordingto the invention to Aβ(1-42) protein fragments whose sequence comprisesthe contiguous amino acids 24 to 42, preferably 22 to 42 and inparticular 20 to 42. According to another aspect, the N-terminalsequences to be removed may be sequences having at least 7, preferablyhaving at least 9 and in particular having at least 11, amino acids ofthe N-terminal sequence of the amyloid β(1-42) protein. Accordingly,preference is given according to the invention in particular tofragments of the amyloid β(1-42) protein which are N-terminallytruncated by from 7 to 23, preferably 9 to 21 and in particular 11 to19, amino acids. These fragments correspond to the formula A β(x42),where x is from 8 to 24, preferably 10 to 22 and in particular 12 to 20.According to the invention, the A β(x42) fragments are thereforepreferably to be selected from among the following fragments: Aβ(8-42),Aβ(9-42), Aβ(10-42), Aβ(11-42), Aβ(12-42), Aβ(13-42), Aβ(14-42),Aβ(15-42), Aβ(16-42), Aβ(17-42), Aβ(18-42), Aβ(19-42), Aβ(20-42),Aβ(21-42), Aβ(22-42), A β(23-42) and Aβ(24-42). Special fragments arethe amyloid β(20-42) fragment obtainable by the action of thermolysinand the amyloid β(12-42) fragment obtainable by the action of GluCendoproteinase.

Derivatives of the invention are also those forms which are derived froman amyloid β(1-42) protein which has 1 or 2 further C-terminal aminoacids. Examples thereof thus include oligomers of the Aβ(143) protein orderivatives thereof corresponding to the comments above.

The inventive method of preparing the oligomers may essentially comprisethree steps, the first of which is optional but advantageous, the secondstep is absolutely required for preparation of the oligomers A and B andthe third step serves to prepare the oligomers B of the invention.

Step 1 relates to unfolding of the protein. For this purpose, hydrogenbond-breaking agents such as, for example, hexafluoroisopropanol (HFIP)may be allowed to act on the protein. Times of action of a few minutes,for example about 10 to 60 minutes, are sufficient when the temperatureof action is from about 20 to 50° C. and in particular about 35 to 40°C. Subsequent dissolution of the residue evaporated to dryness,preferably in concentrated form, in suitable organic solvents misciblewith aqueous buffers, such as, for example, dimethyl sulfoxide (DMSO),results in a suspension of the at least partially unfolded protein,which can be used in step 2 of the method of the invention. If required,the stock suspension may be stored at low temperature, for example atabout −20° C., for an interim period.

Alternatively to step 1 above, the protein may be taken up in slightlyacidic, preferably aqueous, solution, for example an about 10 mM aqueousHCl solution. After an incubation time of usually a few minutes,insoluble components are removed by centrifugation. A few minutes at 10000 g is expedient. These method steps are preferably carried out atroom temperature, i.e. a temperature in the range from 20 to 30° C. Thesupernatant obtained after centrifugation contains the amyloid β(1-42)protein and may be stored at low temperature, for example at about −20°C., for an interim period.

Step 2 relates to oligomerization of the protein to give the oligomersA. For this purpose, a detergent is allowed to act on the, optionally,at least partially unfolded protein until sufficient oligomer A has beenproduced.

Preference is given to using ionic detergents, in particular anionicdetergents.

According to a particular embodiment, a detergent of the formula (I):R—X,is used, in which the radical R is unbranched or branched alkyl havingfrom 6 to 20 and preferably 10 to 14 carbon atoms or unbranched orbranched alkenyl having from 6 to 20 and preferably 10 to 14 carbonatoms, the radical X is an acidic group or salt thereof, with X beingpreferably selected from among -COO⁻M⁺, -SO₃ ^(−M) ⁺, and especially-OSO₃ ⁻M⁺and M⁺ is a hydrogen cation or an inorganic or organic cationpreferably selected from alkali metal and alkaline earth metal cationsand ammonium cations.

Advantageous are detergents of the formula (I), in which R is unbranchedalkyl of which alk−1-yl radicals must be mentioned in particular.Particular preference is given to sodium dodecyl sulfate (SDS). Lauricacid and oleic acid can also be used advantageously. The sodium salt ofthe detergent lauroylsarcosin (also known as sarkosyl NL−30 or Gardol®)is also particularly advantageous.

The time of detergent action in particular depends on whether—and ifyes, to what extent—the protein subjected to oligomerization hasunfolded. If, according to step 1, the protein has been treatedbeforehand with a hydrogen bond-breaking agent, i.e. in particular withhexafluoroisopropanol, times of action in the range of a few hours,advantageously from about 1 to 20 and in particular from about 2 to 10hours, are sufficient when the temperature of action is about 20 to 50°C. and in particular about 35 to 40° C. If a less unfolded or anessentially not unfolded protein is the starting point, correspondinglylonger times of action are expedient. If the amyloid β(1-42) protein hasbeen pretreated, for example, according to the procedure indicated aboveas an alternative to step 1 or said protein is directly introduced tostep 2, times of action in the range from about 5 to 30 hours and inparticular from about 10 to 20 hours are sufficient when the temperatureof action is about 20 to 50° C. and in particular about 35 to 40° C.After incubation, insoluble components are advantageously removed bycentrifugation. A few minutes at 10 000 g is expedient.

The detergent concentration to be chosen depends on the detergent used.If SDS is used, a concentration in the range from 0.01 to 1% by weight,preferably from 0.05 to 0.5% by weight, for example of about 0.2% byweight, proves expedient. If lauric acid or oleic acid are used,somewhat higher concentrations are expedient, for example in a rangefrom 0.05 to 2% by weight, preferably from 0.1 to 0.5% by weight, forexample of about 0.5% by weight.

The detergent action should take place at a salt concentrationapproximately in the physiological range. Thus, in particular NaClconcentrations in the range from 50 to 500 mM, preferably from 100 to200 mM and particularly at about 140 mM are expedient.

The resulting solution containing the oligomers A, i.e. in particularoligomers A1 and/or A2, can be stored at low temperature, for example atabout −20° C., for an interim period. Said solution may be subjected assuch to the use of the invention or to further reaction to give theoligomers B, or further work-up or purification steps follow first whichachieve in particular further concentration of at least one of theoligomers A of the invention. In particular, it is possible to removethe oligomers of the invention by means of chromatographic methods fromother protein components present in the solution and in particular fromthose derived from the amyloid β(1-42) protein. Particularly suitablefor this purpose are affinity chromatography methods which may employ,for example, specific antibodies, i.e. in particular also theoligomer-specific antibodies of the invention.

Step 3 relates to oligomerization to give the oligomers B. Preferably, acomposition containing oligomer A is chosen as a reactant for this step.In this respect, the oligomers A are, according to the invention,important intermediates for the preparation of oligomers B. If saidcomposition is from step 2, it regularly contains detergent and a saltconcentration in the physiological range. It is then expedient to reducedetergent action and salt concentration. This may be carried out byreducing the concentration of detergent and salt, for example, bydiluting, expediently with water or a buffer of lower saltconcentration, for example Tris-HCl, pH 7.3. Dilution factors in therange from about 2 to 10, advantageously in the range from about 3 to 8and in particular of about 4, have proved suitable. The reduction indetergent action may also be achieved by adding substances which canneutralize said detergent action. Examples of these include substancescapable of complexing the detergents, like substances capable ofstabilizing cells in the course of purification and extraction measures,for example particular EO/PO block copolymers, in particular the blockcopolymer under the trade name Pluronic® F 68. Alkoxylated and, inparticular, ethoxylated alkyl phenols such as the ethoxylatedt-octylphenols of the Triton® X series, in particular Triton® X100,3-(3-cholamidopropyldimethylammonio)−1-propanesulfonate (CHAPS®) oralkoxylated and, in particular, ethoxylated sorbitan fatty esters suchas those of the Tween® series, in particular Tween® 20, in concentrationranges around or above the particular critical micelle concentration.

Subsequently, the solution is incubated until sufficient oligomer B hasbeen produced. Times of action in the range of several hours, preferablyin the range from about 10 to 30 hours and in particular in the rangefrom about 15 to 25 hours, are sufficient when the temperature of actionis about 20 to 50° C. and in particular about 35 to 40° C. The solutionmay then be concentrated and possible residues may be removed bycentrifugation. Here too, a few minutes at 10 000 g proves expedient.The supernatant obtained after centrifugation contains oligomers B.

The resulting solution containing the oligomers B, i.e. in particularthe oligomers B1 and/or B2, may be stored at low temperature, forexample at about −80° C., for an interim period. Said solution may besubjected as such to the use of the invention or further work-up orpurification steps may follow first. Regarding this, reference is madeto the comments above on corresponding measures in connection with theoligomers A.

Further work-up or purification steps may also be carried out for thepurpose of essentially completely removing the detergent used forpreparing the oligomers. For example, the oligomers B may first beprecipitated from the detergent-containing solution, isolated andredissolved in detergent-free medium. Measures for precipitatingproteins are sufficiently known to the skilled worker. According to theinvention, the addition of aqueous-methanolic acetic acid has provedexpedient. Without being bound to a particular mechanism, the detergentor variation of detergent and salt concentrations seems to put theprotein in defined soluble forms which clearly differ from the proteinstarting form dissolved in aqueous physiological buffers, as can bedetected, for example, by means of denaturing or native gelelectrophoresis or gel permeation chromatography. This is astonishing,since detergents are normally capable of disaggregating, i.e.disassembling to subunits, protein aggregates, whereas, according to theinvention, defined oligomers are obtained starting from a monomer proneto aggregation.

Derivatives of oligomers of the invention are expediently prepared bycarrying out the above-described method of the invention on alreadyappropriately derivatized amyloid β(1-42) protein. Alternatively, it isalso possible to derivative the oligomer but this should not alter thestructure of said oligomer. Suitable protein-chemical measures are knownto the skilled worker.

Crosslinking of oligomers of the invention or derivatives thereof may becarried out in a manner known per se. If, for example, glutardialdehydeis used as crosslinker, a solution resulting from method step 2 or 3 ofthe invention can be treated with a glutardialdehyde solution. After afew hours at room temperature, the oligomers will have reacted with theglutardialdehyde. The reaction may then be stopped in a manner known perse by reacting the excess glutardialdehyde with reagents generally knownfor this purpose, such as ethanolamine. Depending on whether oligomers Aor B of the invention are crosslinked, a solution of crosslinkedoligomers of the invention referred to as A-CL and B-CL, respectively,is obtained.

It is possible, in particular for the optionally crosslinked oligomers Bor derivatives thereof, following the synthesis thereof, to increaseagain the salt concentration, without impairing the stability of saidoligomers. This is important with regard to the use of said oligomers,for example in the case that for the use physiological conditions areexpedient (cellular applications, in vivo applications).

Oligomers of fragments of the amyloid β(1-42) protein may be prepared inprinciple either starting from corresponding monomer fragments by6ligomerization or starting from oligomers of said amyloid β(1-42)protein by proteolysis. Thus, according to the second method variant, asolution resulting from method steps 2 or 3 of the invention can betreated with protease. When the desired degree of proteolysis isreached, the protease is inactivated in a generally known manner. Theresulting oligomers may then be isolated following the proceduresalready described herein and, if required, processed further by furtherwork-up and purification steps.

The oligomers of the invention and the compositions comprising them aredistinguished by their homogeneity and stability. They are soluble inphysiological media, for example physiological saline, and differ fromfibrillar forms by their rather globular appearance. They have theadvantage of having a spatial structure which is distinctly differentfrom other Aβ(1-42) forms, in particular the monomer, nontoxicoligomers, the Aβ(1-42)—comprising precursor molecule APP, protofibrilsand fibrils. They are therefore particularly suitable for generatingspecific antibodies both in vivo and in vitro and make possible, forexample, a specific active immunization. In this connection, it may becrucial to use for immunization only those oligomers which bring on thedisease. Other forms of Aβ(1-42), such as the monomeric molecule orsmaller oligomers, may be necessary for important signal functions inthe organism. Likewise, removal of the fibrillar depositions which arepresumably important for cell lining may damage the organism.

In the same way, it is possible to implement possible therapies usingthe homogeneous oligomers of the invention and compositions containingsaid oligomers, such as passive immunization, the use of destabilizersof the oligomeric forms and the use of receptor (partial) agonists orantagonists. Thus a homogeneous oligomer preparation makes also possiblea specific production of polyclonal or monoclonal antibodies for passiveimmunization. It is also possible to find receptor molecules or signalmolecules which are relevant to the disease and which are influenced bysaid oligomeric form by using the oligomers of the invention andcompositions containing these.

Owing to their involvement in amyloid β protein-associated physiologicalprocesses, the oligomers of the invention have diagnostic andtherapeutic value. Thus the present invention relates to the use ofoligomers of the invention and of derivatives thereof, optionally in theform of a corresponding composition, in diagnostic in vitro and invivo-detection methods.

Amyloid β protein-associated physiological processes include thoseassociated with depositions of amyloid proteins (amyloidoses). Theseinclude both those processes which result in structural modifications ofnerve tissue and are important, for example, in Alzheimer's disease andDown's syndrome and those processes which affect other tissues, such asamyloid microangiopathies, for example congophilic amyloid angiopathy(CAA).

The present invention further relates to the use of oligomers of theinvention and of derivatives thereof, optionally in the form of acorresponding composition, for generating oligomer-specific antibodies.

Here, the inventive oligomers based on truncated forms of the Aβ(1-42)protein are of particular importance: due to the missing N terminus,they can generate a distinctly more selective immune response than theAβ(1-42) protein. While the highly immunogenic N terminus in theclassical Aβ(1-42) protein predominantly produces antibodies which arespecific for this region of the molecule, for example the antibodies6E10 (F. Signet) and BAM-10 (Sigma, St. Louis) which, according to themanufacturers' information, are directed against the N terminus (6E10:Aβ(1-17) and BAM-10 Aβ(1-12)), the antibodies obtainable using theoligomers of the invention based on truncated Aβ(1-42) forms recognizeoligomer-specific regions, thereby achieving a selectivity compared toother Aβ(1 -42) forms. This advantage may be utilized in particular foractive vaccination, since the more selective immune response resultingafter vaccination (versus APP, monomeric forms, protofibrils, fibrils,plaques) also entails fewer side effects (e.g. brain hemorrhages,impairment of physiological neurotrophic activity of the in situmonomeric form). Superior production and selection of, in particular,monoclonal antibodies for passive immunization, i.e. an antibodytherapy, are also possible.

One aspect of this use is the generation of oligomer-specific antibodieswithin the framework of a therapy.

The present invention therefore further relates to the use of anoligomer of the invention or of a derivative thereof in the therapeuticfield, in particular as vaccine.

Such a vaccine is usually a pharmaceutical composition which comprisesat least one oligomer of the invention and/or at least one derivativethereof of the invention. For this purpose, it is possible to use inparticular any of the compositions of the invention which include two ormore oligomers or a combination of different compositions. Thus it ispossible to carry out a vaccination using in particular the oligomers B,i.e. B1 and B2, or derivatives thereof. The composition may furthercomprise a physiologically suitable carrier and, optionally, furtherexcipients, for example immunostimulants.

While in principle any suitable carriers may be chosen, the type ofcarrier usually depends on the route of administration. Thus thevaccines of the invention may be formulated in particular in a formsuitable for parenteral, for example intravenous, intramuscular andsubcutaneous administration. In these cases, the carrier preferablyincludes water, saline, alcohol, a fat, a wax and/or a buffer.

It is possible to use any of a multiplicity of immunostimulants in thevaccines of the invention. For example, an adjuvant may be included.Most adjuvants contain a substance which ought to protect the antigenfrom rapid catabolism, such as aluminum hydroxide or a mineral oil, andalso a protein derived from lipid A, Bortadella pertussis orMycobacterium tuberculosis. Suitable adjuvants are usually commerciallyavailable, for example complete or incomplete Freund's adjuvant; AS-2;aluminum salts such as aluminum hydroxide (as gel, where appropriate) oraluminum phosphate; calcium salts, iron salts or zinc salts; aninsoluble suspension of acylated tyrosine; acylated sugars; cationicallyor anionically derivatized polysaccharides; polyphosphazenes;biologically degradable microspheres; monophosphoryl lipid A. Cytokinessuch as GM-CSF or Interleukin-2, -7 or -12 may likewise be used asadjuvants.

The present invention furthermore relates to a method of producingantibodies, which comprises

-   -   i) immunizing a host with at least one oligomer of the        invention, derivative thereof or composition; and    -   ii) obtaining an antibody-containing host serum produced as a        response to said immunization.

According to a particular embodiment of the method of producing theantibodies, the immunization is carried out by administeringimmunization cocktails comprising mixtures of various oligomers oroligomer derivatives of the invention. In particular, it may beexpedient to administer in the course of said method a plurality ofimmunization cocktails whose oligomer composition differs.

If the oligomers or oligomer derivatives to be used are not or onlyweakly immunogenic, their immunogenicity may be increased by couplingthem to carriers, preferably to a carrier protein such as keyhole limpethemocyanin (KLH), Limulus polyphenus hemocyanin (LPH), bovine serumalbumin (BSA) or ovalbumin (OVA). For this purpose, there are a numberof commonly known possible couplings available to the skilled worker. Apossible expedient example is the reaction with glutardialdehyde, forexample by incubating the oligomer or oligomer mixture with a suitablepeptide or peptide mixture in water or an aqueous solvent. This reactionmay conveniently be carried out at ambient temperature, i.e. usuallyroom temperature. However, it may also be expedient to reduce orslightly increase the temperature. The reaction usually produces thedesired result within a few hours, a reaction time of 2 h being withinthe-usual range, for example. The glutardialdehyde concentration isusually in the ppm to % range, expediently from 10 ppm up to 1%,preferably from 10 ppm to 0.5%. Optimization of the reaction parametersis within the artisan's skills and should take into account that theoligomers A and B or oligomer derivatives are stable under the chosenreaction conditions.

The immunization cocktails are prepared by first combining thecomponents to be used. It is of advantage to incubate the resultingcomponent mixture initially. This is conveniently carried out at ambienttemperature, i.e. usually at room temperature. However, it may beexpedient to cool or slightly heat said mixture. The incubation periodis usually from a few minutes to a few hours, with an incubation time of1 h having proved advantageous.

Immunization cocktails contain, in addition to the antigen, usuallyfurther excipients, in particular adjuvants commonly used forimmunization, for example Freund's adjuvant. More specifically, completeFreund's adjuvant is used for the first immunization, whereas anyfurther immunizations are carried out with incomplete Freund's adjuvant.The immunization cocktail is prepared by adding the antigen (immunogen),preferably in the form of the above-described component mixture, to theexcipient(s), with the antigen usually being emulsified.

Suitable hosts are in particular rodents or else rabbits. These or othersuitable hosts are injected with the immunization cocktails, preferablysubcutaneously. The antibody titers may be determined using animmunoassay, for example competitively using a sheep antiserum directedagainst host IgG and a labeled oligomer. Thus it may be decided towardthe end of immunization whether a particular host is suitable forproducing antibodies. If, for example, four immunizations are carriedout, it is possible to determine the antibody titer after the thirdimmunization and then to obtain antibodies from animals having asufficient antibody titer.

The antibodies produced are preferably obtained by taking blood from thehosts over a period of several weeks or months. Finally, the host can bebled. Serum containing the desired antibodies may be obtained from theblood obtained in a manner known per se. The whole serum thus obtainedmay, if required, be further purified by the skilled worker in order toconcentrate the antibody fraction present therein and in particular theoligomer-recognizing antibodies.

According to a particular embodiment of this method, at least oneantibody of the serum is selected, which antibody specificallyrecognizes the oligomer used as immunogen or a derivative thereof or atleast one oligomer or derivative thereof present in the composition usedas immunogen. In this context, specificity means a higher bindingaffinity of the antibody for the immunogen than for other, in particularrelated proteins, especially in comparison with monomeric amyloidβ(1-42) protein and also with oligomeric or multimeric amyloid β(1-42)protein aggregates having a higher molecular weight than the oligomersof the invention. It is also possible to obtain in this mannermonoclonal oligomer-specific antibodies. To this end, however,preference is given to removing from the hosts spleen tissue and,starting from the thus obtained spleen lymphocytes, to establish in theusual manner hybridomas which produce the monoclonal antibodies.

DETAILED DESCRIPTION OF ANTIBODY PRODUCTION

B lymphocytes which, in totality, contain an antibody repertoirecomposed of hundreds of millions of different antibody specificities arepart of the mammalian immune system. A normal immune response to aparticular antigen means selection of one or more antibodies of saidrepertoire which specifically bind to said antigen, and the success ofan immune response is based at least partially on the ability of saidantibodies to specifically recognize (and ultimately to eliminate) thestimulating antigen and to ignore other molecules in the environment ofsaid antibodies.

The usefulness of antibodies which specifically recognize one particulartarget antigen has led to the development of monoclonal antibodytechnology. Standardized hybridoma technology now allows the productionof antibodies with a single specificity for an antigen of interest. Morerecently, recombinant antibody techniques such as in-vitro screening ofantibody libraries have been developed. These techniques likewise allowantibodies having a single specificity for an antigen of interest to beproduced.

In the method of the invention, the antigen of interest may be allowedto act on the antibody repertoire either in vivo or in vitro.

According to one embodiment, the antigen is allowed to act on therepertoire by immunizing an animal in vivo with said antigen. Thisin-vivo approach may furthermore comprise establishing from thelymphocytes of an animal a number of hybridomas and selecting ahybridoma which secretes an antibody specifically binding to saidantigen. The animal to be immunized may be, for example, a mouse, rat,rabbit, chicken, camelid or sheep or may be a transgenic version of anyof the animals mentioned above, for example a transgenic mouse withhuman immunoglobulin genes, which produces human antibodies after anantigenic stimulus. Other types of animals which may be immunizedinclude mice with severe combined immunodeficiency (SCID) which havebeen reconstituted with human peripheral mononuclear blood cells(chimeric hu-PBMC SCID mice) or with lymphoid cells or precursorsthereof, as well as mice which have been treated with a lethal totalbody irradiation, then protected against radiation with bone marrowcells from a mouse with severe combined immunodeficiency (SCID) andsubsequently transplanted with functional human lymphocytes (the“Trimera” system). Another type of an animal to be immunized is ananimal (e.g. a mouse) in whose genome an endogenous gene encoding theantigen of interest has been switched off (knocked out), for example byhomologous recombination, so that, after immunization with the antigen,said animal recognizes said antigen as foreign. It is obvious to theskilled worker that the polyclonal or monoclonal antibodies produced bythis method are characterized and selected by using known screeningmethods which include, but are not limited to, ELISA techniques.

According to another embodiment, the antigen is allowed to act on theantibody repertoire in vitro by screening a recombinant antibody librarywith said antigen. The recombinant antibody library may be expressed,for example, on the surface of bacteriophages or on the surface of yeastcells or on the surface of bacterial cells. In a variety of embodiments,the recombinant antibody library is an scFv library or an Fab library,for example. According to another embodiment, antibody libraries areexpressed as RNA-protein fusions.

Another approach to producing antibodies of the invention comprises acombination of in vivo and in vitro approaches. For example, the antigenmay be allowed to act on the antibody repertoire by immunizing an animalin vivo with said antigen and then screening in vitro with said antigena recombinant antibody library prepared from lymphoid cells of saidanimal or a single domain antibody library (e.g. containing heavy and/orlight chains). According to another approach, the antigen is allowed toact on the antibody repertoire by immunizing an animal in vivo with saidantigen and then subjecting a recombinant antibody library or singledomain library produced from lymphoid cells of said animal to affinitymaturation. According to another approach, the antigen is allowed to.act on the antibody repertoire by immunizing an animal in vivo with saidantigen, then selecting individual antibody-producing cells secreting anantibody of interest and obtaining from said selected cells cDNAs forthe variable region of the heavy and light chains (e.g. by means of PCR)and expressing said variable regions of the heavy and light chains inmammalian host cells in vitro (this being referred to as selectedlymphocyte antibody method or SLAM), thereby being able to furtherselect and manipulate the selected antibody gene sequences. Moreover,monoclonal antibodies may be selected by expression cloning byexpressing the antibody genes for the heavy and light chains inmammalian cells and selecting those mammalian cells which secrete anantibody having the desired binding affinity.

Accordingly, one aspect of the present invention is to provide definedantigens for screening and counter screening. Thus it is possible,according to the invention, to select those polyclonal and monoclonalantibodies which bind an oligomer of the invention or derivative thereofbut not other forms of the Aβ(1-42) protein, APP, amyloid fibrils oramyloid plaques and a number of other nonrelated antigens and tissues.

It is sufficiently known to the skilled worker that antibody selectionsare based on well-defined antigens. In contrast, less well-definedantigens are not selective enough when used. In short, the acting andselecting in vitro is similar to affinity chromatography, with “ligands”for the desired antigen being removed from those which bind the antigenwith insufficient affinity. The degree of concentration of the desiredantibodies from the huge pool of other antibodies is therefore a directconsequence of the quality of the antigen. Surprisingly, the oligomersof the invention and derivatives thereof are antigens which can be usedto concentrate suitable, relevant and selective antibodies andefficiently remove them from antibodies which recognize other formsassociated with the Aβ(1-42) protein and other nonrelated antigens.

The methods of the invention for producing antibodies can be used toproduce various types of antibodies. These include essentially humanantibodies, chimeric antibodies, humanized antibodies and CDR graftantibodies and also antigen-binding moieties thereof.

Methods of producing antibodies of the invention are described below. Adistinction is made here between in-vivo approaches, in-vitro approachesor a combination of both.

In-vivo Approaches

Starting from the in-vivo generated antibody-producing cells, monoclonalantibodies may be produced by means of standardized techniques such asthe hybridoma technique originally described by Kohler and Milstein(1975, Nature 256:495497) (see also Brown et al. (1981) J Immunol127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int J. Cancer 29:269-75).The technology of producing monoclonal antibody hybridomas issufficiently known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36).Briefly, an immortalized cell line (typically a myeloma) is fused withlymphocytes (typically splenocytes or lymph node cells or peripheralblood lymphocytes) of a mammal immunized with the oligomer of theinvention or derivative thereof, and the culture supernatants of theresulting hybridoma cells are screened in order to identify a hybridomawhich produces a monoclonal antibody with specificity for the oligomerof the invention or for a derivative thereof. Any of the many well knownprotocols for fusing lymphocytes and immortalized cell lines can beapplied for this purpose (see also G. Galfre et al. (1977) Nature266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lemer, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the skilled worker will appreciate that there arediverse variations of such methods, which are likewise useful.Typically, the immortalized cell line (e.g. a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas may be established by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the invention with animmortalized mouse cell line. Preferred immortalized cell lines aremouse myeloma cell lines which are sensitive to culture mediumcontaining hypoxanthine, aminopterin and thymidine (HAT medium). Any ofa number of myeloma cell lines may be used standardwise as fusionpartner, for example the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma cell lines are available from the AmericanType Culture Collection (ATCC), Rockville, MD. Typically, HAT-sensitivemouse myeloma cells are fused to mouse splenocytes using polyethyleneglycol (PEG). Hybridoma cells resulting from the fusion are thenselected using HAT medium, thereby killing unfused and unproductivelyfused myeloma cells (unfused splenocytes die after several days becausethey are not transformed). Hybridoma cells producing monoclonalantibodies which specifically recognize the oligomer of the invention ora derivative thereof are identified by screening the hybridoma culturesupernatants for such antibodies, for example by using a standard ELISAassay in order to select those antibodies which can specifically bindthe oligomer of the invention or a derivative thereof.

Depending on the type of the desired antibody, various host animals maybe used for in-vivo immunization. A host expressing itself an endogenousversion of the antigen of interest may be used. Alternatively, it ispossible to use a host which has been made deficient in an endogenousversion of the antigen of interest. For example, mice which had beenmade deficient in a particular endogenous protein via homologousrecombination at the corresponding endogenous gene (i.e. knockout mice)have been. shown to generate a humoral response to the protein withwhich they have been immunized and therefore to be able to be used forproduction of high-affinity monoclonal antibodies to the protein (see,for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237;Lunn, M. P. et al. (2000) J. Neurochem. 75:404-412).

A multiplicity of nonhuman mammals are suitable hosts for antibodyproduction in order to produce nonhuman antibodies to the oligomer ofthe invention or to a derivative thereof. They include mice, rats,chickens, camelids, rabbits and goats (and knockout versions thereof),although preference is given to mice for the production of hybridoma.Furthermore, a nonhuman host animal expressing a human antibodyrepertoire may be used for producing essentially human antibodies to ahuman antigen with dual specificity. Nonhuman animals of this kindinclude transgenic animals (e.g. mice) bearing human immunoglobulintransgenes (chimeric hu-PBMC SCID mice) and human/mouse irradiationchimeras which are described in more detail below.

According to one embodiment, the animal immunized with an oligomer ofthe invention or derivative thereof is a nonhuman mammal, preferably amouse, which is transgenic due to human immunoglobulin genes so thatsaid nonhuman mammal makes human antibodies upon antigenic stimulation.Typically, immunoglobulin transgenes for heavy and light chains withhuman germ line configuration are introduced into such animals whichhave been altered such that their endogenous heavy and light chain lociare inactive. If such animals are stimulated with antigen (e.g. with ahuman antigen), antibodies derived from the human immunoglobulinsequences (human antibodies) are produced. It is possible to make fromthe lymphocytes of such animals human monoclonal antibodies by means ofstandardized hybridoma technology. For a further description oftransgenic mice with human immunoglobulins and their use in theproduction of human antibodies, see, for example, U.S. Pat. No.5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049(Abgenix Inc.), and U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825,U.S. Pat. No. 5,625,126, US 5,633,425, U.S. Pat. No. 5,661,016, U.S.Pat. No. 5,770,429, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,877,397 andWO 99/45962 (Genpharm Inc.); see also MacQuitty, J. J. and Kay, R. M.(1992) Science 257:1188; Taylor, L. D. et al. (1992) Nucleic Acids Res.20:6287-6295; Lonberg, N. et al. (1994) Nature 368:856-859; Lonberg, N.and Huszar, D. (1995) Int. Rev. Immunol. 13:65-93; Harding, F. A. andLonberg, N. (1995) Ann. N Y. Acad. Sci. 764:536-546; Fishwild, D. M. etal. (1996) Nature Biotechnology 14:845-851; Mendez, M. J. et al. (1997)Nature Genetics 15:146-156; Green, L. L. and Jakobovits, A. (1998) J.Exp. Med. 188:483495; Green, L. L. (1999) J. Immunol. Methods 231:11-23;Yang, X. D. et al. (1999) J. Leukoc. Biol. 66:401410; Gallo, M. L. etal. (2000) Eur. J. Immunol. 30:534-540.

According to another embodiment, the animal which is immunized with anoligomer of the invention or a derivative thereof may be a mouse withsevere combined immunodeficiency (SCID), which has been reconstitutedwith human peripheral mononuclear blood cells or lymphoid cells orprecursors thereof. Such mice which are referred to as chimeric hu-PBMCSCID mice produce human immunoglobulin responses upon antigenicstimulation, as has been proved. For a further description of these miceand of their use for generating antibodies, see, for example, Leader,K.A. et al. (1992) Immunology 76:229-234; Bombil, F. et al. (1996)Immunobiol. 195:360-375; Murphy, W.J. et al. (1996) Semin. Immunol.8:233-241; Herz, U. et al. (1997) Int. Arch. Allergy Immunol.113:150-152; Albert, S.E. et aL (1997) J. Immunol. 159:1393-1403;Nguyen, H. et al. (1997) MicrobioL Immunol. 41:901-907; Arai, K. et al.(1998) J. Immunol. Methods 217:79-85; Yoshinari, K. and Arai, K. (1998)Hybridoma 17:4145; Hutchins, W.A. et al. (1999) Hybridoma 18:121-129;Murphy, W.J. et al. (1999) Clin. Immunol. 90:22-27; Smithson, S.L. etal. (1999) Mol. Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect.Diseases 180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:3545.

According to another embodiment, the animal which is immunized with anoligomer of the invention or a derivative thereof is a mouse which hasbeen treated with a lethal total body irradiation, then protected fromradiation with bone marrow cells from mice with severe combinedimmunodeficiency (SCID) and subsequently transplanted with functionalhuman lymphocytes. This type of chimera, referred to as the Trimerasystem, is used in order to produce human monoclonal antibodies byimmunizing said mice with the antigen of interest and then producingmonoclonal antibodies by using standardized hybridoma technology. For afurther description of these mice and of their use for generatingantibodies, see, for example, Eren, R. et al. (1998) Immunology93:154-161; Reisner, Y and Dagan, S. (1998) Trends Biotechnol.16:242-246; lhan, E. et al. (1999) Hepatology 29:553-562; and Bocher,W.O. et al. (1999) Immunology 96:634-641.

In-vitro Approaches

As an alternative to producing antibodies of the invention byimmunization and selection, antibodies of the invention may beidentified and isolated by screening recombinant combinatorialimmunoglobulin libraries with an oligomer of the invention or derivativethereof to thereby isolate immunoglobulin library members whichspecifically bind to said oligomer or derivative thereof. Kits forgenerating and screening display libraries are commercially available(e.g. the Pharmacia Recombinant Phage Antibody System, catalog No.27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, catalog No.240612). In many embodiments, the display library is an scFv library oran Fab library. The phage display technique for screening recombinantantibody libraries has been adequately described. Examples of methodsand compounds which can be used particularly advantageously forgenerating and screening antibody display libraries can be found, forexample, in McCafferty et al. WO 92/01047, U.S. Pat. No. 5,969,108 andEP 589 877 (describes in particular scFv display), Ladner et al. U.S.Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698,U.S. Pat. No. 5,837,500 and EP 436 597 (describes pill fusion, forexample); Dower et al. WO 91/17271, U.S. Pat. No. 5,427,908, U.S. Pat.No. 5,580,717 and EP 527 839 (describes in particular Fab display);Winter et al. International Publication WO 92/20791 and EP 368,684(describes in particular the cloning of sequences for variableimmunoglobulin domains); Griffiths et al. U.S. Pat. No. 5,885,793 and EP589 877 (describes in particular isolation of human antibodies to humanantigens by using recombinant libraries); Garrard et al. WO 92/09690(describes in particular phage expression techniques); Knappik et al. WO97/08320 (describes the human recombinant antibody library HuCal);Salfeld et al. WO 97/29131, (describes production of a recombinant humanantibody to a human antigen (human tumor necrosis factor alpha) and alsoin-vitro affinity maturation of the recombinant antibody) and Salfeld etal. U.S. Provisional Application No. 60/126,603 and the patentapplications based hereupon (likewise describes production ofrecombinant human antibodies to human antigen (human interleukin-12),and also in-vitro affinity maturation of the recombinant antibody).

Further descriptions of screenings of recombinant antibody libraries canbe found in scientific publications such as Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896;Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS89:3576-3580; Garrard et al. (1991) BioITechnology 9:1373-1377;Hoogenboom et al. (1991) Nuc Acid Res 19:41334137; Barbas et al. (1991)PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; andKnappik et aL (2000) J. Mol. Biol. 296:57-86.

As an alternative to using bacteriophage display systems, recombinantantibody libraries may be expressed on the surface of yeast cells or ofbacterial cells. WO 99/36569 describes methods of preparing andscreening libraries expressed on the surface of yeast cells. WO 98/49286describes in more detail methods of preparing and screening librariesexpressed on the surface of bacterial cells.

Once an antibody of interest of a combinatorial library has beenidentified, the DNAs encoding the light and heavy chains of saidantibody are isolated by means of standardized molecular-biologicaltechniques, for example by means of PCR amplification of DNA from thedisplay package (e.g. the phage) which has been isolated during libraryscreening. Nucleotide sequences of genes for light and heavy antibodychains, which may be used for preparing PCR primers, are known to theskilled worker. A multiplicity of such sequences are described, forexample, in Kabat, E.A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242 and in the database ofsequences of the human germ line VBASE.

An antibody or antibody moiety of the invention may be produced byrecombinantly expressing the genes for light and heavy immunoglobulinchains in a host cell. In order to recombinantly express an antibody, ahost cell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the light and heavy immunoglobulinchains of said antibody, thereby expressing the light and heavy chainsin the host cell and secreting them preferably into the medium in whichsaid host cells are cultured. The antibodies can be isolated from thismedium. Standardized recombinant DNA methods are used in order to obtaingenes for heavy and light antibody chains, to insert said genes intorecombinant expression vectors and to introduce said vectors into hostcells. Methods of this kind are described, for example, in Sambrook,Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al.(eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al

Once DNA fragments encoding VH and VL segments of the antibody ofinterest are obtained, said DNA fragments may be further manipulatedusing standardized recombinant DNA techniques, for example in order toconvert the genes for variable regions to genes for full length antibodychains, to genes for Fab fragments or to an scFv gene. Thesemanipulations comprise linking a VL- or VH-encoding DNA fragmentoperatively to another DNA fragment encoding another protein, forexample a constant antibody region or a flexible linker. The term“operatively linked” is to be understood here as meaning that the twoDNA fragments are linked to one another in such a way that the aminoacid sequences encoded by said two DNA fragments remain in frame.

The isolated DNA encoding the VH region may be converted to a gene for afull length heavy chain by operatively linking the VH-region encodingDNA with another DNA molecule encoding heavy chain constant regions(CH1, CH2 and CH3). The sequences of human heavy chain constant regiongenes are well known (see, for example, Kabat, E.A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),and DNA fragments spanning said regions may be obtained by means ofstandardized PCR amplification. The heavy chain constant region may be aconstant region from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, withpreference being given to a constant region from IgG1 or IgG4. To obtaina gene for a heavy chain Fab fragment, the VH-encoding DNA may beoperatively linked to another DNA molecule encoding merely the heavychain constant region CH1.

The isolated DNA encoding the VL region may be converted to a gene for afull length light chain (and a gene for an Fab light chain) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region CL. The sequences of genes of theconstant region of human light chain are well known (see Kabat, E. A.,et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242), and DNA fragments spanning said regions may be obtained bymeans of standardized PCR amplification. The light chain constant regionmay be a constant kappa or lambda region, a constant kappa region beingpreferred.

In order to generate an scFv gene, the VH- and VL-encoding DNA fragmentsmay be operatively linked to another fragment encoding a flexiblelinker, for example the amino acid sequence (Gly₄-Ser)₃ so that the VHand VL sequences are expressed as a continuous single-chain protein,with the VL and VH regions being linked to one another via said flexiblelinker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature(1990) 348:552-554).

Single domain VH and VL with specificity for an oligomer of theinvention or for a derivative thereof may be isolated from single domainlibraries by the above-described methods. Two VH single-domain chains(with or without CHl) or two VL chains or a pair of one VH chain and oneVL chain with the desired specificity may be used for removing oligomersof the invention or derivatives thereof from the body.

In order to express the recombinant antibodies or antibody moieties ofthe invention, the DNAs encoding partial or full length light and heavychains may be inserted into expression vectors so as to operatively linkthe genes to transcriptional and translational control sequences. Inthis context, the term “operatively linked” is to be understood asmeaning that an antibody gene is ligated in a vector in such a way thattranscriptional and translational control sequences within the vectorfulfill their intended function of regulating transcription andtranslation of said antibody gene.

The expression vector and the expression control sequences are chosen soas to be compatible with the expression host cell used. The gene for theantibody light chain and the gene for the antibody heavy chain may beinserted into separate vectors or both genes are inserted into the sameexpression vector, this being the usual case. The antibody genes areinserted into the expression vector by means of standardized methods(for example ligation of complementary restriction cleavage sites on theantibody gene fragment and the vector, or ligation of blunt ends, if norestriction cleavage sites are present). The expression vector mayalready carry sequences for antibody constant regions prior to insertionof the sequences for the light and heavy chains. For example, oneapproach is to convert the VH and VL sequences to full length antibodygenes by inserting them into expression vectors already encoding theheavy and, respectively, light chain constant regions, therebyoperatively linking the VH segment to the CH segment(s) within thevector and also operatively linking the VL segment to the CL segmentwithin the vector. Additionally or alternatively, the recombinantexpression vector may encode a signal peptide which facilitatessecretion of the antibody chain from the host cell. The gene for saidantibody chain may be cloned into the vector, thereby linking the signalpeptide in frame to the N terminus of the gene for the antibody chain.The signal peptide may be an immuno-globulin signal peptide or aheterologous signal peptide (i.e. a signal peptide from anon-immunoglobulin protein). In addition to the genes for the antibodychain, the expression vectors of the invention may have regulatorysequences controlling expression of the genes for the antibody chain ina host cell. The term “regulatory sequence” is intended to includepromoters, enhancers and further expression control elements (e.g.polyadenylation signals) which control transcription or translation ofthe genes for the antibody chain. Regulatory sequences of this kind aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, CA (1990). The skilledworker will appreciate that the expression vector design which includesselection of regulatory sequences may depend on factors such as thechoice of host cell to be transformed, the desired strength ofexpression of the protein, etc. Preferred regulatory sequences forexpression in mammalian host cells include viral elements resulting in astrong protein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), simian virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus (e.g. the adenovirus major late promoter(AdMLP)) and polyoma. For a further description of viral regulatoryelements and sequences thereof, see, for example, U.S. Pat. No.5,168,062 to Stinski, U.S. Pat. No. 4,510,245 to Bell et al. and U.S.Pat. No. 4,968,615 to Schaffner et aL

Apart from the genes for the antibody chain and the regulatorysequences, the recombinant expression vectors of the invention may haveadditional sequences such as those which regulate replication of thevector in host cells (e.g. origins of replication) and selectable markergenes. The selectable marker genes facilitate the selection of hostcells into which the vector has been introduced (see, for example, U.S.Pat Nos. 4,399,216, 4,634,665 and 5,179,017, all to Axel et al.). Forexample, it is common for the selectable marker gene to render a hostcell into which the vector has been inserted resistant to drugs such asG418, hygromycin or methotrexate. Preferred selectable marker genesinclude the gene for dihydrofolate reductase (DHFR) (for use in dhfrhost cells with methotrexate selection/ amplification) and the neo gene(for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding said heavy and light chains is(are) transfected into a hostcell by means of standardized techniques. The various forms of the term“transfection” are intended to comprise a multiplicity of techniquescustomarily used for introducing exogenous DNA into a prokaryotic oreukaryotic host cell, for example electroporation, calcium phosphateprecipitation, DEAE-dextran transfection, and the like. Although it istheoretically possible to express the antibodies of the invention eitherin prokaryotic or eukaryotic host cells, preference is given toexpressing the antibodies in eukaryotic cells and, in particular, inmammalian host cells, since the probability of a correctly folded andimmunologically active antibody being assembled and secreted is higherin such eukaryotic cells and in particular mammalian cells than inprokaryotic cells. Prokaryotic expression of antibody genes has beenreported as being ineffective for production of high yields of activeantibody (Boss, Mass. and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing recombinant antibodies ofthe invention include CHO cells (including dhfr CHO cells described inUrlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:42164220, whichare used together with a DHFR-selectable marker, as described, forexample, in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenintroducing recombinant expression vectors encoding the antibody genesinto mammalian host cells, the antibodies are produced by culturing thehost cells until the antibody is expressed in said host cells or,preferably, the antibody is secreted into the culture medium in whichthe host cells grow. The antibodies may be isolated from the culturemedium by using standardized protein purification methods.

It is likewise possible to use host cells in order to produce moietiesof intact antibodies, such as Fab fragments or scFv molecules.Variations of the above-described procedure are of course included inthe invention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of the invention. If either light or heavy chainsare present which are not required for binding of the antigen ofinterest, then the DNA encoding either such a light or such a heavychain or both may be removed partially or completely by means ofrecombinant DNA technology. Molecules expressed by such truncated DNAmolecules are likewise included in the antibodies of the invention. Inaddition, it is possible to produce bifunctional antibodies in which aheavy chain and a light chain are an antibody of the invention and theother heavy chain and the other light chain have specificity for anantigen different from the antigen of interest, by crosslinking anantibody of the invention to a second antibody by means of standardizedchemical methods.

In a preferred system for recombinant expression of an antibody of theinvention or an antigen-binding moiety thereof, a recombinant expressionvector encoding both the antibody heavy chain and the antibody lightchain is introduced into dhfr CHO cells by means of calciumphosphate-mediated transfection. Within the recombinant expressionvector, the genes for the heavy and light antibody chains are in eachcase operatively linked to regulatory CMV enhancer/AdMLP-promoterelements in order to effect strong transcription of said genes. Therecombinant expression vector also carries a DHFR gene which can be usedfor selecting CHO cells transfected with the vector by usingmethotrexate selection/amplification. The selected transformed hostcells are cultured so that the heavy and light antibody chains areexpressed, and intact antibody is isolated from the culture medium.Standardized molecular-biological techniques are used in order toprepare the recombinant expression vector, to transfect the host cells,to select the transformants, to culture said host cells, and to obtainthe antibody from the culture medium. Thus the invention relates to amethod of synthesizing a recombinant antibody of the invention byculturing a host cell of the invention in a suitable culture mediumuntil a recombinant antibody of the invention has been synthesized. Themethod may furthermore comprise isolating said recombinant antibody fromsaid culture medium.

As an alternative to screening recombinant antibody libraries by phagedisplay, other methods known to the skilled worker may be used forscreening large combinatorial libraries to identify the antibodies ofthe invention. In one type of an alternative expression system, therecombinant antibody library is expressed in the form of RNA-proteinfusions, as described in WO 98/31700 to Szostak and Roberts, and inRoberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, in-vitro translation of synthetic mRNAscarrying on their 3′ end puromycin, a peptidyl acceptor antibiotic,generates a covalent fusion of an mRNA and the peptide or proteinencoded by it. Thus a specific mRNA of a complex mixture of mRNAs (e.g.a combinatorial library) may be concentrated on the basis of theproperties of the encoded peptide or protein (e.g. of the antibody or amoiety thereof), such as binding of said antibody or said moiety thereofto an oligomer of the invention or a derivative thereof. Nucleic acidsequences which encode antibodies or moieties thereof and which areobtained by screening of such libraries may be expressed by recombinantmeans in the above-described manner (e.g. in mammalian host cells) andmay, in addition, be subjected to further affinity maturation by eitherscreening in further rounds mRNA-peptide fusions, introducing mutationsinto the originally selected sequence(s), or using other methods ofin-vitro affinity maturation of recombinant antibodies in theabove-described manner.

Combinations of In-vivo and In-vitro Approaches

The antibodies of the invention may likewise be produced by using acombination of in-vivo and in-vitro approaches such as methods in whichan oligomer of the invention or a derivative thereof is first allowed toact on an antibody repertoire in a host animal in vivo to stimulateproduction of oligomer- or derivative-binding antibodies and thenfurther antibody selection and/or antibody maturation (i.e.optimization) are accomplished with the aid of one or more in-vitrotechniques. According to one embodiment, a combined method of this kindmay comprise firstly immunizing a nonhuman animal (e.g. a mouse, rat,rabbit, chicken, camelid, goat or a transgenic version thereof or achimeric mouse) with said oligomer of the invention or derivativethereof to stimulate an antibody response to the antigen and thenpreparing and screening a phage display antibody library by usingimmunoglobulin sequences of lymphocytes which have been stimulated invivo by the action of said oligomer or derivative. The first step ofthis combined procedure may be carried out in the manner described abovein connection with the in-vivo approaches, while the second step of thisprocedure may be carried out in the manner described above in connectionwith the in-vitro approaches. Preferred methods of hyperimmunizingnonhuman animals with subsequent in-vitro screening of phage displaylibraries prepared from said stimulated lymphocytes include thosedescribed by BioSite Inc., see, for example, WO 98/47343, WO 91/17271,U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.

According to another embodiment, a combined method comprises firstlyimmunizing a nonhuman animal (e.g. a mouse, rat, rabbit, chicken,camelid, goat or a knockout and/or transgenic version thereof, or achimeric mouse) with an oligomer of the invention or derivative thereofto stimulate an antibody response to said oligomer or derivative thereofand selecting the lymphocytes which produce the antibodies having thedesired specificity by screening hybridomas (prepared, for example, fromthe immunized animals). The genes for the antibodies or single domainantibodies are isolated from the selected clones (by means ofstandardized cloning methods such as reverse transcriptase polymerasechain reaction) and subjected to in-vitro affinity maturation in orderto improve thereby the binding properties of the selected antibody orthe selected antibodies. The first step of this procedure may beconducted in the manner described above in connection with the in-vivoapproaches, while the second step of this procedure may be conducted inthe manner described above in connection with the in-vitro approaches,in particular by using methods of in-vitro affinity maturation, such asthose described in WO 97/29131 and WO 00/56772.

In a further combined method, the recombinant antibodies are generatedfrom individual isolated lymphocytes by using a procedure which is knownto the skilled worker as selected lymphocyte antibody methods (SLAM) andwhich is described in U.S. Pat. No. 5,627,052, WO 92/02551 and Babcock,J.S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In thismethod, a nonhuman animal (e.g. a mouse, rat, rabbit, chicken, camelid,goat, or a transgenic version thereof, or a chimeric mouse) is firstlyimmunized in vivo with an oligomer of the invention or a derivativethereof to stimulate an immune response to said oligomer or derivative,and then individual cells secreting antibodies of interest are selectedby using an antigen-specific hemolytic plaque assay. To this end, theoligomer or derivative thereof or structurally related molecules ofinterest may be coupled to sheep erythrocytes, using a linker such asbiotin, thereby making it possible to identify individual cellssecreting antibodies with suitable specificity by using the hemolyticplaque assay. Following the identification of cells secreting antibodiesof interest, cDNAs for the variable regions of the light and heavychains are obtained from the cells by reverse transcriptase PCR, andsaid variable regions may then be expressed in association with suitableimmunoglobulin constant regions (e.g. human constant regions) inmammalian host cells such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences derived from invivo-selected lymphocytes may then be subjected to further in-vitroanalysis and in-vitro selection by spreading out the transfected cells,for example, in order to isolate cells expressing antibodies with thedesired specificity. The amplified immunoglobulin sequences mayfurthermore be manipulated in vitro.

Analogously to the above-described procedures, it is also possible toprepare antibodies having specificity for an oligomer of the inventionor derivative thereof and for structurally related synthetic molecules.This comprises

-   -   i) providing an antigen which comprises a structural feature        shared by the oligomer of the invention or derivative thereof        and the structurally related synthetic molecules;    -   ii) exposing an antibody repertoire to said antigen; and    -   iii) selecting from said repertoire an antibody which binds to        two structurally related molecules, thereby obtaining the        antibody having the desired specificity.

The present invention relates to the oligomer-specific antibodiesobtainable by the above methods as well as to the use thereof forpreparing a medicament for the treatment of amyloid 1-associateddementing disorders or for preparing a composition for diagnosingamyloid B-associated dementing disorders.

The antibodies obtainable according to the invention include inparticular antisera which can be obtained by the above methods. Saidantisera may be whole sera, i.e. blood obtained from the host afterremoving the cellular and coagulable components, or fractions of saidserum which contain in particular a concentrated immunoglobulin fractionand preferably a concentrated, oligomer-recognizing immunoglobulinfraction. Fractions of this kind may be obtained using the methodsdescribed above in connection with antibody purification.

The antisera of the invention are polyclonal, i.e. they containantibodies of different specificity, usually of different classes andsubclasses, normally all L-chain isotypes are represented and multipleprotein epitopes are recognized.

When using various oligomers of the invention as immunogens, then theantisera of the invention are usually cross-reactive.

According to another aspect, the antibodies obtainable according to theinvention also include monoclonal antibodies, in particular chimeric andhumanized antibodies, and also oligomer-binding fragments thereof.

The present invention relates to proteins and in particular toantibodies binding to an oligomer of the invention or derivativethereof, i.e. antibodies having specificity for an oligomer of theinvention or derivative thereof. The present invention also relates tomoieties of said proteins or antibodies, in particular toantigen-binding moieties thereof, i.e. protein or antibody moietiesbinding to an oligomer of the invention or derivative thereof.

The antibody of the invention is preferably chosen so as to haveparticular binding kinetics (e.g. high affinity, low dissociation, lowoff rate, strong neutralizing activity) for the specific binding to anoligomer of the invention or a derivative thereof.

Thus preference is given to proteins and, in particular, antibodieshaving an affinity for the oligomer of the invention or derivativethereof in the range of K_(D)=10⁻⁸−10⁻¹² M. Particular preference isgiven to high-affinity proteins and in particular antibodies bindingwith an affinity greater than K_(d)=10⁻⁸ M, with an affinity greaterthan K_(d)=10⁻⁹ M, with an affinity greater than K_(d)=10⁻¹⁰ M or withan affinity greater than K_(d)=10⁻¹¹ M.

According to another aspect, preference is given to those proteins and,in particular, antibodies which bind other Aβ(1-42) forms, in particularmonomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein withcomparatively lower affinity, in particular with lower affinity thanK_(d)=10⁻⁸ M.

Accordingly, preference is given according to the invention especiallyto those proteins and, in particular, antibodies which bind the oligomeror derivative thereof with higher affinity than monomeric Aβ(1 -42)protein and/or monomeric Aβ(1 -40) protein. Particular preference isgiven to affinity ratios of 10, 100 or 1000.

According to another aspect, the antibodies of the invention may bechosen so as to bind the oligomer or derivative thereof with a koff rateconstant of 0.1s⁻¹ or less. Increasing preference is given to rateconstants k_(off) of 1×10⁻²s⁻¹ or less, 1×10⁻³s⁻¹ or less, 1×10⁻⁴s⁻¹ orless, or 1×10⁻⁵s⁻¹ or less, in the order indicated.

Furthermore, antibodies of the invention may be chosen so as to inhibitthe activity, in particular the neurotoxic activity, of oligomers of theinvention or derivatives thereof with an IC₅₀ of 1×10⁻⁶ M or less.Increasing preference is given to inhibition constants IC₅₀ of 1×10⁻⁷ Mor less, 1×10 ⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁰ M or less, or1×10⁻¹¹ M or less, in the order indicated.

The antibodies are preferably isolated antibodies. According to anotheraspect, the antibodies are neutralizing antibodies. The antibodies ofthe invention include monoclonal and recombinant antibodies. Accordingto a multiplicity of embodiments, the antibody may comprise an aminoacid sequence derived entirely from a single species, such as a humanantibody or a mouse antibody. According to other embodiments, theantibody may be a chimeric antibody or a CDR graft antibody or anotherform of a humanized antibody.

The term “antibody” is intended to refer to immunoglobulin moleculesconsisting of 4 polypeptide chains, two heavy (H) chains and two light(L) chains. The chains are usually linked to one another via disulfidebonds. Each heavy chain is composed of a variable region of said heavychain (abbreviated here as HCVR or VH) and a constant region of saidheavy chain. The heavy chain constant region consists of three domainsCH1, CH2 and CH3. Each light chain is composed of a variable region ofsaid light chain (abbreviated here as LCVR or VL) and a constant regionof said light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region consists of three CDRs and four FRs whichare arranged from the N terminus to the C terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “antigen-binding moiety” of an antibody (or simply “antibodymoiety”) refers to one or more fragments of an antibody havingspecificity for an oligomer of the invention or derivative thereof, saidfragment(s) still being capable of specifically binding said oligomer orderivative thereof. Fragments of a complete antibody have been shown tobe able to carry out the antigen-binding function of an antibody. Inaccordance with the term “antigen-binding moiety” of an antibodyexamples of binding fragments include (i) an Fab fragment, i.e. amonovalent fragment composed of the VL, VH, CL and CH1 domains; (ii) anF(ab′)₂ fragment, i.e. a bivalent fragment comprising two Fab fragmentslinked to one another in the hinge region via a disulfide bridge; (iii)an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragmentcomposed of the FL and VH domains of a single arm of an antibody; (v) adAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VHdomain or of VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolatedcomplementarity-determining region (CDR). Although the two domains ofthe Fv fragment, namely VL and VH, are encoded by separate genes, theymay further be linked to one another using a synthetic linker andrecombinant methods, making it possible to prepare them as a singleprotein chain in which the VL and VH regions combine in order to formmonovalent molecules (known as single chain Fv (ScFv); see, for example,Bird et al. (1988) Science 242:423426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). The term “antigen-binding moiety” ofan antibody is also intended to comprise such single chain antibodies.Other forms of single chain antibodies such as “diabodies” are likewiseincluded here. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker which is too short for the two domains being able to combine onthe same chain, thereby forcing said domains to pair with complementarydomains of a different chain and to form two antigen-binding sites (see,for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Furthermore, an antibody or antigen-binding moiety thereof may be partof a larger immunoadhesion molecule formed by covalent or noncovalentassociation of said antibody or antibody moiety with one or more furtherproteins or peptides. Relevant to such immunoadhesion molecules are theuse of the streptavidin core region in order to prepare a tetramericscFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies andHybridomas 6:93-101) and the use of a cystein residue, a marker peptideand a C-terminal polyhistidine tag in order to produce bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody moieties such as Fab and F(ab′)₂fragments may be produced from whole antibodies by using conventionaltechniques such as digestion with papain or pepsin. In addition,antibodies, antibody moieties and immunoadhesion molecules may beobtained by using standardized recombinant DNA techniques. An “isolatedantibody having specificity for an oligomer of the invention orderivative thereof” means an antibody having specificity for an oligomerof the invention or derivative thereof, which is essentially free ofother antibodies having different antigen specificities, i.e. inparticular an antibody which is free of antibodies specifically bindingto other forms of the Aβ(1-42) protein, as described above.

The term “neutralizing antibody” means an antibody whose binding to aparticular antigen results in the inhibition of the biological activityof said antigen. Said inhibition of the biological activity of theantigen may be assessed by measuring one or more indicators for saidbiological activity of the antigen, using a suitable in-vitro or in-vivoassay.

The term “monoclonal antibody” means an antibody derived from ahybridoma (e.g. an antibody secreted by a hybridoma prepared by means ofhybridoma technology such as the standardized hybridoma methodsaccording to Kohler and Milstein). An antibody which is derived from ahybridoma and which has specificity for an oligomer of the invention orderivative thereof is therefore referred to as monoclonal antibody.

The term “recombinant antibody” refers to antibodies which are produced,expressed, generated or isolated by recombinant means, such asantibodies which are expressed using a recombinant expression vectortransfected into a host cell; antibodies isolated from a recombinantcombinatorial antibody library; antibodies isolated from an animal (e.g.a mouse) which is transgenic due to human immunoglobulin genes (see, forexample, Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295); orantibodies which are produced, expressed, generated or isolated in anyother way in which particular.immunoglobulin gene sequences (such ashuman immunoglobulin gene sequences) are assembled with other DNAsequences. Recombinant antibodies include, for example, chimeric, CDRgraft and humanized antibodies.

The term “human antibody” refers to antibodies whose variable andconstant regions correspond to or derive from immunoglobulin sequencesof the human germ line, as described, for example, by Kabat et al. (seeKabat, et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242). However, the human antibodies of the inventionmay contain amino acid residues not encoded by human germ lineimmunoglobulin sequences (for example mutations which have beenintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs, and in particular in CDR3.Recombinant human antibodies of the invention have variable regions andmay also contain constant regions derived from immunoglobulin sequencesof the human germ line (see Kabat, E.A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). According toparticular embodiments, however, such recombinant human antibodies aresubjected to in-vitro mutagenesis (or to a somatic in-vivo mutagenesis,if an animal is used which is transgenic due to human Ig sequences) sothat the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences which although related to orderived from VH and VL sequences.of the human germ line, do notnaturally exist in vivo within the human antibody germ line repertoire.According to particular embodiments, recombinant antibodies of this kindare the result of selective mutagenesis or back mutation or of both.

The term “back mutation” refers to a method which comprises replacingsome of or all of the somatically mutated amino acids of a humanantibody with the corresponding germ line residues of a homologous germline antibody sequence. The sequences for the heavy and light chains ofa human antibody of the invention are compared separately with the germline sequences in the VBASE database to identify the sequences havingthe highest homology. Imprints on the human antibodies of the inventionare reverted to the germ line sequence by mutating defined nucleotidepositions encoding such deviating amino acids. The direct or indirectimportance of each amino acid identified in this way as a candidate fora back mutation for antigen binding should be investigated, and an aminoacid impairing a desired property of said human antibody after mutationshould not be incorporated in the final human antibody. In order to keepthe number of amino acids for back mutation as low as possible, thoseamino acid positions which, although deviating from the closest germline sequence, are identical to the corresponding amino acid sequence ofa second germ line sequence may remain unchanged, provided that saidsecond germ line sequence is identical and colinear with the sequence ofthe human antibody of the invention in at least 10 and preferably in 12amino acids on both sides of the amino acid in question. Back mutationsmay be carried out at any stage of antibody optimization.

The term “chimeric antibody” refers to antibodies which containsequences for the variable region of the heavy and light chains from onespecies but in which the sequences of one or more of the CDR regions ofVH and/or VL have been replaced with CDR sequences of another species,such as antibodies having variable regions of the heavy and light chainsfrom mouse, in which one or more of the mouse CDRs (e.g. CDR3) have beenreplaced with human CDR sequences.

The term “humanized antibody” refers to antibodies which containsequences of the variable region of heavy and light chains from anonhuman species (e.g. mouse, rat, rabbit, chicken, camelid, goat) butin which at least one part of the VH and/or VL sequence has been alteredin order to be more “human-like”, i.e. to be more similar to variablesequences of the human germ line. One type of a humanized antibody is aCDR graft antibody in which human CDR sequences have been inserted intononhuman VH and VL sequences to replace the corresponding nonhuman CDRsequences.

One way of measuring the binding kinetics of an antibody is by means ofsurface plasmon resonance. The term “surface plasmon resonance” refersto an optical phenomenon by which biospecific interactions can beanalyzed by detecting changes in protein concentrations by means of abiosensor matrix, using, for example, the BlAcore system (PharmaciaBiosensor AB, Uppsala, Sweden and Piscataway, N.J.). For furtherdescriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26;Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., etal. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991)Anal. Biochem. 198:268-277.

The term “K_(off)” refers to the off rate constant for the dissociationof an antibody from the antibody/antigen complex.

The term “K_(d)” refers to the dissociation constant of a particularantibody-antigen interaction.

The binding affinity of the antibodies of the invention may be evaluatedby using standardized in-vitro immunoassays such as ELISA or BlAcoreanalyses.

Apart from antibodies, the protein may be a T cell receptor-derivedmolecule or a T cell receptor-derived receptor domain or a fusionprotein of said receptor domain with an Fc moiety of an immunoglobulin.

The present invention also relates to pharmaceutical agents(compositions) containing a protein of the invention and in particularan antibody of the invention and also, optionally, a pharmaceuticallysuitable carrier. Pharmaceutical compositions of the invention mayfurthermore contain at least one additional therapeutic agent, forexample one or more additional therapeutic agents for the treatment of adisease for whose relief the antibodies of the invention are useful. If,for example, the antibody of the invention binds to an oligomer of theinvention, the pharmaceutical composition may furthermore contain one ormore additional therapeutic agents useful for the treatment of disordersin which the activity of said oligomer is important.

Pharmaceutically suitable carriers include any solvents, dispersingmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption-delaying agents, and the like, as long as they arephysiologically compatible. Pharmaceutically acceptable carriersinclude, for example, water, saline, phosphate-buffered saline,dextrose, glycerol, ethanol and the like, and combinations thereof. Inmany cases, preference is given to using isotonic agents, for examplesugars, polyalcohols such as mannitol or sorbitol, or sodium chloride inaddition. Pharmaceutically suitable carriers may furthermore containrelatively small amounts of auxiliary substances such as wetting agentsor emulsifiers, preservatives or buffers, which increase the half lifeor efficacy of the antibodies.

The pharmaceutical compositions may be suitable for parenteraladministration, for example. Here, the antibodies are preparedpreferably as injectable solutions with an antibody content of 0.1 - 250mg/ml. The injectable solutions may be prepared in liquid or lyophilizedform, the dosage form being a flint glass or vial, an ampoule or afilled syringe. The buffer may contain L-histidine (1 - 50 mM,preferably 5 - 10 mM) and have a pH of 5.0 - 7.0, preferably of 6.0.Further suitable buffers include, without being limited thereto, sodiumsuccinate, sodium citrate, sodium phosphate or potassium phosphatebuffers. Sodium chloride may be used in order to adjust the tonicity ofthe solution to a concentration of 0 - 300 mM (preferably 150 mM for aliquid dosage form). Cryoprotectants, for example sucrose (e.g. 0 - 10%,preferably 0.5 - 1.0%) may also be included for a lyophilized dosageform. Other suitable cryoprotectants are trehalose and lactose. Fillers,for example mannitol (e.g. 1 - 10%, preferably 2 - 4%) may also beincluded for a lyophilized dosage form. Stabilizers, for exampleL-methionine (e.g. 51 - 50 mM, preferably 5 - 10 mM) may be used both inliquid and lyophilized dosage forms. Further suitable fillers areglycine and arginine. Surfactants, for example polysorbate 80 (e.g. 0 -0.05%, preferably 0.005 - 0.01%), may also be used. Further surfactantsare polysorbate 20 and BRIJ surfactants.

The compositions of the invention may have a multiplicity of forms.These include liquid, semisolid and solid dosage forms, such as liquidsolutions (e.g. injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended type of administration and on thetherapeutic application. Typically, preference is given to compositionsin the form of injectable or infusible solutions, for examplecompositions which are similar to other antibodies for passiveimmunization of humans. The preferred route of administration isparenteral (e.g. intravenous, subcutaneous, intraperitoneal,intramuscular). According to a preferred embodiment, the antibody isadministered by intravenous infusion or injection. According to anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions must typically be sterile and stable under thepreparation and storage conditions. The compositions may be formulatedas solution, micro-emulsion, dispersion, liposome or another orderedstructure suitable for high active substance concentrations. Sterileinjectable solutions may be prepared by introducing the active compound(i.e. the antibody) in the required amount into a suitable solvent,where appropriate with one or a combination of the abovementionedingredients, as required, and then sterile-filtering said solution.Dispersions are usually prepared by introducing the active compound intoa sterile vehicle containing a basic dispersion medium and, whereappropriate, other required ingredients. In the case of a sterilelyophilized powder for preparing sterile injectable solutions, vacuumdrying and spray drying are preferred methods of preparation, whichproduces a powder of the active ingredient and, where appropriate, offurther desired ingredients from a previously sterile-filtered solution.The correct flowability of a solution may be maintained by using, forexample, a coating such as lecithin, by maintaining, in the case ofdispersions the required particle size or by using surfactants. Aprolonged absorption of injectable compositions may be achieved byadditionally introducing into the composition an agent which delaysabsorption, for example monostearate salts and gelatin.

The antibodies of the invention may be administered by a multiplicity ofmethods known to the skilled worker, although the preferred type ofadministration for many therapeutic applications is subcutaneousinjection, intravenous injection or infusion. The skilled worker willappreciate that the route and/or type of administration depend on theresult desired. According to particular embodiments, the active compoundmay be prepared with a carrier which protects the compound against rapidrelease, such as, for example, a formulation with controlled release,which includes implants, transdermal plasters and microencapsulatedrelease systems. Biologically degradable biocompatible polymers such asethylene vinyl acetate, polyanhydrides, polyglycol acid, collagen,polyorthoesters and polylactic acid may be used. The methods ofpreparing such formulations are well known to the skilled worker, see,for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. According toparticular embodiments, an antibody of the invention may be administeredorally, for example in an inert diluent or an assimilable ediblecarrier. The antibody (and further ingredients, if desired) may also beenclosed in a hard or soft gelatin capsule, compressed to tablets oradded directly to food. For oral therapeutic administration, theantibodies may be mixed with excipients and used in the form ofswallowable tablets, buccal tablets, capsules, elixirs, suspensions,syrups and the like. If it is intended to administer an antibody of theinvention via a route other than the parenteral one, it may be necessaryto choose a coating from a material which prevents its inactivation.

The antibodies of the invention are preferably capable of neutralizing,both in vitro and in vivo, the activity of oligomers of the invention orderivatives thereof to which they bind. Said antibodies may therefore beused for inhibiting the activity of oligomers of the invention orderivatives thereof, for example in a cell culture containing saidoligomers or derivatives thereof or in human individuals or othermammals in which said oligomers or derivatives thereof are present.According to one embodiment, the invention relates to a method ofinhibiting the activity of oligomers of the invention or derivativesthereof, which method comprises allowing an antibody of the invention toact on an oligomer or derivative thereof so as to inhibit the activityof said oligomer or derivative thereof. Said activity may be inhibitedin vitro, for example. For example, the antibody of the invention may beadded to a cell culture which contains or is suspected to contain theoligomer of the invention or derivative thereof, in order to inhibit theactivity of said oligomer or derivative thereof in said culture.Alternatively, the activity of the oligomer or derivative thereof may beinhibited in an individual in vivo.

Thus the present invention further relates to a method.of inhibiting theactivity of oligomers of the invention or derivatives thereof in anindividual who suffers from a disorder in which the amyloid B protein isinvolved and in which in particular the activity of said oligomer of theinvention or derivative thereof is important. Said method comprises theadministration of at least one antibody of the invention to theindividual with the aim of inhibiting the activity of the oligomer orderivative thereof to which the antibody binds. Said individual ispreferably a human being. An antibody of the invention may beadministered for therapeutic purposes to a human individual. Inaddition, an antibody of the invention may be administered to a nonhumanmammal for veterinary purposes or within the framework of an animalmodel for a particular disorder. Such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention (forexample for testing dosages and time courses of administration).

Disorders in which the oligomers of the invention or derivatives thereofplay a part include in particular disorders in whose development and/orcourse an oligomer of the invention or derivative thereof is involved.These are in particular those disorders in which oligomers of theinvention or derivatives thereof are evidently or presumably responsiblefor the pathophysiology of said disorder or are a factor whichcontributes to the development and/or course of said disorder.Accordingly, those disorders are included here in which inhibition ofthe activity of oligomers of the invention or derivatives thereof canrelieve symptoms and/or progression of the disorder. Such disorders canbe verified, for example, by an increased concentration of oligomers ofthe invention or derivatives thereof in a biological fluid of anindividual suffering from a particular disorder (e.g. increasedconcentration in serum, plasma, CSF, urine, etc.). This may be detected,for example, by using an antibody of the invention. The oligomers of theinvention and derivatives thereof play an important part in thepathology associated with a multiplicity of disorders in whichneurodegenerative elements, cognitive deficits, neurotoxic elements andinflammatory elements are involved.

The antibodies of the invention may be administered together with one ormore additional therapeutic agents which are useful in the treatment ofthe above-described disorders.

The pharmaceutical compositions of the present invention usually containa therapeutically active amount or a prophylactically active amount ofat least one antibody of the invention. Depending on the treatmentdesired, for example whether a therapeutic or prophylactic treatment isdesired, dosage plans can be chosen and adapted. For example, a singledose, multiple separate doses distributed over time or an increasing ordecreasing dosage may be administered, depending on the requirements ofthe therapeutic situation. It is particularly advantageous to formulateparenteral compositions in single dosage form in order to facilitateadministration and to ensure uniformity of the dosage.

A therapeutically or prophylactically active amount of an antibody ofthe invention may be, for example, in the range of 0.1 - 20 mg/kg andpreferably 1 - 10 mg/kg, without being limited thereto. These amountsmay, of course, vary, depending on the type and severity of thecondition to be relieved.

Within the framework of diagnostic usage of the antibodies, qualitativeor quantitative specific oligomer determination serves in particular todiagnose disease-relevant amyloid β(1-42) forms. In this context,specificity means the possibility of being able to detect a particularoligomer or oligomer mixture with sufficient sensitivity. The antibodiesof the invention advantageously have sensitivities of less than 10 ng/mlof sample, preferably of less than 1 ng/ml of sample and particularlypreferably of less than 100 pg/ml of sample, meaning that at least theconcentration of oligomer per ml of sample, indicated in each case,advantageously also lower concentrations, can be detected by theantibodies of the invention.

The determination is carried out immunologically. This may be carriedout in principle by using any analytical or diagnostic assay method inwhich antibodies are used, including agglutination and precipitationtechniques, immunoassays, immunohistochemical methods and immunoblottechniques, for example Western blotting or dot blot methods. In vivomethods, for example imaging methods, are also included here.

The use in immunoassays is advantageous. Suitable are both competitiveimmunoassays, i.e. antigen and labeled antigen (tracer) compete forantibody binding, and sandwich immunoassays, i.e. binding of specificantibodies to the antigen is detected by a second, usually labeledantibody. These assays may be either homogeneous, i.e. withoutseparation into solid and liquid phases, or heterogeneous, i.e. boundlabels are separated from unbound ones, for example via solidphase-bound antibodies. Depending on labeling and method of measurement,the various heterogeneous and homogeneous immunoassay formats can beclassified into particular classes, for example RIAs(radioimmunoassays), ELISA (enzyme-linked immunosorbent assay), FIA(fluorescence immunoassay), LIA (luminescence immunoassay), TRFIA(time-resolved FIA), IMAC (immunoactivation), EMIT (enzynie-multipliedimmune test), TIA (turbodimetric immunoassay), I-PCR (immuno-PCR).

For the oligomer determination of the invention, preference is given tocompetitive immunoassays in which labeled oligomer (tracer) competeswith the oligomer to be quantified of the sample for binding to theantibody used. The amount of antigen, i.e. the amount of oligomer, inthe sample can be determined from the amount of the displaced tracerwith the aid of a standard curve.

Of the labels available for these purposes, enzymes have provedadvantageous. Systems based on peroxidases, in particular horseradishperoxidase, alkaline phosphatase and βD-galactosidase, may be used, forexample. Specific substrates whose conversion can be monitoredphotometrically, for example, are available for these enzymes. Suitablesubstrate systems are based on p-nitrophenyl phosphate (p-NPP),5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NPT),Fast-Red/naphthol-AS-TS phosphate for alkaline phosphatase;2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPT), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 3-dimethylamino-benzoic acid(DMAB) and 3-methyl-2-benzothiazolinehydrazone (MBTH) for peroxidases;o-nitrophenyl-βD-galactoside (o-NPG), p-nitrophenyl-βD-galactoside and4-methylumbelliphenyl-βD-galactoside (MUG) for βD-galactosidase. In manycases, these substrate systems are commercially available in aready-to-use form, for example in the form of tablets which may alsocontain further reagents such as appropriate buffers and the like.

The tracers used are labeled oligomers. In this sense, a particularoligomer can be determined by labeling the oligomer to be determined andusing it as tracer.

The coupling of labels to oligomers for preparing tracers may be carriedout in a manner known per se. The comments above on derivatization ofoligomers of the invention are referred to by analogy. In addition, anumber of labels appropriately modified for conjugation to proteins areavailable, for example biotin-, avidin-, extravidin- orstreptavidin-conjugated enzymes, maleimide-activated enzymes and thelike. These labels may be reacted directly with the oligomer or, ifrequired, with the appropriately derivatized oligomer to give thetracer. If, for example, a streptavidin-peroxidase conjugate is used,then this firstly requires biotinylation of the oligomer. This appliescorrespondingly to the reverse order. For this purpose too, suitablemethods are known to the skilled worker.

If a heterogeneous immunoassay format is chosen, the antigen-antibodycomplex may be separated by binding it to the support, for example viaan anti-idiotypical antibody coupled to said support, e.g. an antibodydirected against rabbit IgG. Supports, in particular microtiter platescoated with appropriate antibodies are known and partly commerciallyavailable.

The present invention further relates to immunoassay sets having atleast one above-described antibody and further components. Said setsare, usually in the form of a packaging unit, a combination of means forcarrying out an oligomer determination of the invention. For the purposeof handling which is as easy as possible, said means are preferablyprovided in an essentially ready-to-use form. An advantageousarrangement offers the immunoassay in the form of a kit. A kit usuallycomprises multiple containers for separate arrangement of components.All components may be provided in a ready-to-use dilution, as aconcentrate for diluting or as a dry substance or lyophilisate fordissolving or suspending; individual or all components may be frozen orstored at room temperature until use. Sera are preferably shock-frozen,for example at −20° C. so that in these cases an immunoassay has to bekept preferably at temperatures below freezing prior to use.

Further components supplied with the immunoassay depend on the type ofsaid immunoassay. Usually, standard protein, tracer which may or may notbe required and control serum are supplied together with the antiserum.Furthermore, microtiter plates, preferably antibody-coated, buffers, forexample for testing, for washing or for conversion of the substrate, andthe enzyme substrate itself may also be included. General principles ofimmunoassays and generation and use of antibodies as auxiliaries inlaboratory and hospital can be found, for example, in Antibodies, ALaboratory Manual (Harlow, E., and Lane, D., Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988).

Furthermore of interest are also substances which inhibit aggregation ofthe oligomers of the invention or accelerate disaggregation thereof.Such substances particularly represent possible therapeutic agents forthe treatment of the above amyloid β-associated disorders such as, forexample, Alzheimer's disease. The present invention therefore alsorelates to a method of characterizing a substance or a substancemixture, which method comprises

-   -   i) providing said substance or said substance mixture in a        suitable manner;    -   ii) allowing said substance or said substance mixture to act on        at least one oligomer of the invention, derivative thereof or a        composition; and    -   iii) determining whether said substance or particular parts of        said substance mixture bind to said oligomer, derivative thereof        or to at least one oligomer or derivative thereof present in        said composition.

Said method may also be carried out on mixtures of biological origin,for example a cell preparation and cell extracts, in order to identifynatural binding partners with affinity for the oligomers of theinvention, for example cell surface antigens, in particular receptors,and soluble ligands, for example particular proteins and mediators.

In addition to mere binding of the substances, further interactions withand influences on the oligomers of the invention may also be a subjectof said method. Thus it is possible to determine, in particular, whether

-   -   the substance is capable of modulating, in particular        inhibiting, the aggregation of amyloid β protein to the        oligomers of the invention;    -   the substance is capable of modulating, in particular promoting,        disaggregation of the oligomers of the invention;    -   the oligomers of the invention cause functional changes in a        binding partner, for example have an agonistic, partially        agonistic, antagonistic or inverse agonistic effect on a        receptor.

Said methods are usually in vitroscreening methods which can be used toselect from a multiplicity of different substances those which appear tobe most promising with respect to a future application. It is possible,for example, to establish by means of combinatorial chemistry extensivesubstance libraries comprising myriads of potential active substances.The screening of combinatorial substance libraries for substances havingthe desired activity can be automated. Screening robots serve toefficiently analyze the individual assays which are preferably arrangedon microtiter plates. Thus the present invention also relates toscreening methods, i.e. both primary and secondary screening methods, inwhich preferably at least one of the methods described below is applied.If several methods are applied, they may be applied to one and the samesample with a time shift or simultaneously or to different samples of asubstance to be tested.

A particularly effective technique for carrying out such methods is thescintillation proximity assay, SPA for short, which is known in thefield of drug screening. Kits and components for carrying out this assaymay be obtained commercially, for example from Amersham PharmaciaBiotech. In principle, solubilized or membrane-bound receptors areimmobilized on small fluoromicrospheres containing a scintillatingsubstance. When, for example, a radioligand binds to the immobilizedreceptors, said scintillating substance is stimulated and emits light,due to the spatial proximity of scintillating substance and radioligand.

Another particularly effective technique for carrying out methods ofthis kind is the FlashPlateR technique known in the field of drugscreening. Kits and components for carrying out this assay may becommercially obtained, for example from NENR Life Science Products. Thisprinciple is likewise based on microtiter plates (96- or 384-well)coated with scintillating substance.

The present invention relates to substances or parts of substancemixtures, which are identifiable according to this method as a ligandbinding to the otigomer, derivative thereof or to at least one oligomeror derivative thereof present in a corresponding composition, as well asto the use thereof for preparing a medicament for the treatment ofamyloid β-associated, in particular dementing, disorders or forpreparing a composition for diagnosing amyloid β-associated, inparticular dementing, disorders.

The following examples are intended to illustrate the invention, withoutlimiting its scope.

In the drawings:

FIG. 1 shows an SDS PAGE of an Aβ(1-42) oligomer A preparation (lane A);an Aβ(1-42) oligomer B preparation (lane B); of standard proteins(molecular marker proteins, lane C);

FIG. 2 shows an SDS PAGE of an Aβ(1-42) oligomer A preparation (lane A);an Aβ(1-42) oligomer A-CL preparation (lane A′); an Aβ(1-42) oligomer Bpreparation (lane B); an Aβ(1-42) oligomer B-CL preparation (lane B′);of standard proteins (molecular marker proteins, lane C);

FIG. 3 shows an SDS PAGE of a biotin Aβ(1-42) oligbmer B preparation(lane A); of standard proteins (molecular marker proteins, lane B);

FIG. 4 shows an SDS PAGE of a fluorescein A β(1-42) oligomer Bpreparation (lane A); of standard proteins (molecular marker proteins,lane B);

FIG. 5 shows a gel permeation chromatography of a solution containingAβ(1-42) lyophilisate in comparison with a preparation containingAβ(1-42) oligomers B;

FIG. 6 shows a NATIVE PAGE of an Aβ(1-42) oligomer B preparation (laneA); of standard proteins (molecular marker proteins, lane B);

FIG. 7 shows the binding of (A) monomeric Aβ(1-42) protein and (B) ofthe Aβ(1-42) oligomers A and (C) Aβ(1-42) oligomers B to the surface ofthe human neuroblastoma cell line IMR-32;

FIG. 8 shows the neurotoxic effect in % after treatment of murinecortical neurons with Aβ(1-42) oligomers B, the error bar correspondingto the 95% confidence interval;

FIG. 9 shows an SDS PAGE of an Aβ(1-42) preparation treated with trypsin(lane 2), chymotrypsin (lane 3), thermolysin (lane 4), elastase (lane5), papain (lane 6) or untreated (lane 7); and of standard proteins(molecular marker proteins, lane 1);

FIG. 10 shows dot blots of the reactivity of 100 pmol (row A); 10 pmol(row B); 1 pmol (row C); 0.1 pmol (row D) and 0.01 pmol (row E); of Aβ(1-42) oligomer B preparation of example 6b (column 1); of HFIP-treatedAβ(1-42) monomer of example 1a (column 2); of thermolysin-cleavedAβ(1-42) oligomer B preparation of example 15a (column 3); ofglutaraldehyde-crosslinked Aβ(1-42) oligomer B preparation of example14a (column 4); of ADDL prepared according to M. P. Lambert et al., J.Neurochem. 79, 595605 (2001) at 4° C. or room temperature or 37° C.(columns 5, 6 and 7, respectively); of Aβ(1-42) dissolved in 0.1% NH₄OH(column 8); of an Aβ(1-42) fibril preparation of example 27 (column 9);and of PBS-diluted APP from Sigma (column 10) with a) the monoclonalantibody 6E10; b) the polyclonal antiserum (d1) of example 25d; c) thepolyclonal antiserum (c1) of example 25c; d) the polyclonal antiserum(al) of example 25a; and e) the polyclonal antiserum (a2) of example25a;

FIG. 11 shows an SDS PAGE of an Aβ(20- 42) oligomer B preparation ofexample 15a (lane B); of an Aβ(12- 42) oligomer B preparation of example15b (lane C); of an Aβ(1-42) oligomer B preparation of example 6b (laneA); and of standard proteins (molecular marker proteins, lane D);

FIG. 12 shows a gel permeation chromatography of the A β(1-42) oligomerB preparation of example 6b in comparison with the Aβ(l1-42) oligomerB-CL preparation of example 14a;

FIG. 13 shows a diagrammatic representation of the monomeric Aβ(1-42)protein (left); of the Aβ(1-42) oligomer (12-mer, A); and of theAβ(12-42) oligomer (12-mer, C) and Aβ(20- 42) oligomer (12-mer, B) bothof which are obtainable by proteolytic cleavage;

FIG. 14 shows the excitatory postsynaptic potential (fEPSP) as afunction of the time of action of Aβ(1-42) oligomers B on hippocampalsections;

FIG. 15 shows an immunofluorescence image depicting the binding ofAβ(I-42) oligomers B to hippocampal rat neurons (a) in comparison withunspecifically bound fluorescence (b).

Unless stated otherwise, concentrations of oligomers of the inventionare expressed in moles of monomeric Aβ(l-42) polypeptide. The term βamyloid(1-42) protein corresponds to the term amyloid β(1-42) protein.Unless stated otherwise, the proteins (polypeptides) used are of humanorigin.

EXAMPLE 1

a) Preparation of a Stock Suspension of Human Aβ(1-42)

2 mg of human B-amyloid(1-42) protein (short name: Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) aredissolved in 800 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubatedin an Eppendorf vessel at 37° C. for 30 min. This is followed byevaporation to dryness in a vacuum concentrator (Speed Vac). The residueis taken up with 88 μl of DMSO, resulting in a 5 mM Aβ(1-42) stocksuspension which can be stored at −20° C.

b) Preparation of a Stock Suspension or Rat Aβ(1 -42)

2 mg of rat β-amyloid(1-42) protein (short name: rat Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) bydissolving 800 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated inan Eppendorf vessel at 37° C. for 30 min. This is followed byevaporation to dryness in a vacuum concentrator (Speed Vac). The residueis taken up with 88 μl of DMSO, resulting in a 5 mM rat Aβ(1-42) stocksuspension which can be stored at −20° C.

EXAMPLE 2

a) Preparation of Aβ(1-42) Oligomers Having Molecular Weights of 15 kDaand 20 kDa [Aβ(1-42) Oligomers A]; Use of SDS

690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) areadded to 60 μl of the stock solution of example 1 a and the mixture isadjusted to SDS content of 0.2% with 75 ,μl of 2% strength sodiumdodecyl sulfate (SDS) solution. This is followed by 5 hours ofincubation at 37° C. and centrifugation for 10 min at 10 000 g. ThisAβ(1-42) oligomer A preparation (approx. 400 μM Aβ(1-42)) can be storedat −20° C.

b) Preparation of Rat Aβ(1 -42) Oligomers Having Molecular Weights of 15kDa and 20 kDa [Rat Aβ(1-42) Oligomers A]; Use of SDS

690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) areadded to 60 μl of the stock solution of example 1b and the mixture isadjusted to SDS content of 0.2% with 75 μl of 2% strength sodium dodecylsulfate (SDS) solution. This is followed by 5 hours of incubation at 37°C. and centrifugation for 10 min at 10 000 g. This rat Aβ(1-42) oligomerA preparation (approx. 400 μM rat Aβ(l 42)) can be. stored at −20° C.

c) Preparation of Aβ(1-42) Oligomers Having Molecular Weights of 15 kDaand 20 kDa [A(1-42) Oligomers A]; Use of Lauric Acid

690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) areadded to 60 μl of the stock solution of example 1a and the mixture isadjusted to lauric acid content of 0.5% with 75 μl of 5% strength lauricacid solution. This is followed by 5 hours of incubation at 37° C. andcentrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomer Apreparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.

d) Preparation of Aβ(1-42) Oligomers Having Molecular Weights of 15 kDaand 20 kDa [Aβ(1-42) Oligomers A]; Use of Oleic Acid

690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) areadded to 60 μl of the stock solution of example 1a and the mixture isadjusted to oleic acid content of 0.5% with 75 μl of 5% strength oleicacid solution. This is followed by 5 hours of incubation at 37° C. andcentrifugation for 10 min at 10 000 9. This Aβ(1-42) oligomer Apreparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.

e) Preparation of Aβ(1-42) Oligomers Having Molecular Weights of 15 kDaand 20 kDa [Aβ(1-42) Oligomers A]; Use of Lauroylsarcosine

690 ,μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) areadded to 60 μl of the stock solution of example 1a and the mixture isadjusted to lauroylsarcosine content of 0.5% with 75 μl of 5% strengthlauroylsarcosine solution. This is followed by 5 hours of incubation at37° C. and centrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomerA preparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.

EXAMPLE 3

a) Alternative Method of Preparing Aβ(1-42) Oligomers Having MolecularWeights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]

1 mg of human β-amyloid(1-42) protein (short name: Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) is takenup in 220 μl of 10 mM aqueous HCl solution and incubated at roomtemperature for 10 min. Insoluble components are removed bycentrifugation at 10 000 g for 5 min. The supernatant (1 mM Aβ(1-42))contains the Aβ(1-42) protein and is processed further as follows:

9 μl of PBS buffer and 1 μl of 2% strength SDS soloution are added to 1μl of the supernatant and the mixture is incubated at 37° C. for 16 h.The hAβ(1-42) oligomer A preparation (100 μM) can be stored at −20° C.

b) Alternative method of preparing rat Aβ(1-42) oligomers havingmolecular weights of 15 kDa and 20 kDa [rat Aβ(1-42) oligomers Al

1 mg of rat β-amyloid(1-42) protein (short name: rat Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) is takenup in 220 μlI of 10 mM aqueous HCl solution and incubated at roomtemperature for 10 min. Insoluble components are removed bycentrifugation at 10 000 g for 5 min. The supernatant (1 mM ratAβ(1-42)) contains the rat amyloid-pfi(1-42) protein and is processedfurther as follows:

9 ;li of PBS buffer and 1 μl of 2% strength SDS soloution are added to 1i of the supernatant and the mixture is incubated at 37° C. for 16 h.The rat Aβ(1-42) oligomer A preparation (100 μM) can be stored at −20°C.

EXAMPLE 4

a) Alternative Method of Preparing Aβ(1-42) Oligomers Having MolecularWeights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]

1 mg of human β-amyloid(1-42) protein (short name: Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) isdissolved in 44 μl of 1% SDS/H₂O (5 mM Aβ(1-42)). 5 μl of the solutionare admixed with 40 μl PBS and 5 μl of 2% SDS and incubated at 37° C.for 16 h. Insoluble components are removed by centrifugation at 10 000 gfor 5 min. The thus obtained Aβ(1-42) oligomer A preparation (500 μMAβ(1-42)) can be stored at −20° C.

b) Alternative Method of Preparing Rat Aβ(1-42) Oligomers HavingMolecular Weights of 15 kDa and 20 kDa [Rat Aβ(1-42) Oligomers A]

1 mg of rat β-amyloid(1-42) protein (short name: rat Aβ(1-42);peptide-synthetic material, lyophilisate, from Bachem, Germany) isdissolved in 44 μl of 1% SDS/H₂O (5 mM rat Aβ(1-42)). 5 μl of thesolution are admixed with 40 μl PBS and 5 μl of 2% SDS and incubated at37° C. for 16 h. Insoluble components are removed by centrifugation at10 000 g for 5 min. The thus obtained rat Aβ(1-42) oligomer Apreparation (500 μM rat Aβ(1-42)) can be stored at −20° C.

EXAMPLE 5

a) Preparation of Aβ(1-42) Oligomers Having Molecular Weights of 38 kDaand 48 kDa [Aβ(1-42) Oligomers B]

An Aβ(1-42) oligomer A solution obtained according to example 2a isdiluted with 2.475 ml of water (0.05% SDS content, 0.1 mM Aβ(1-42)) andincubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42) oligomer Bpreparation can be frozen at −80° C. and stored for further studies.

b) Preparation of Rat Aβ(1-42) Oligomers Having Molecular Weights of 38kDa and 48 kDa (Rat Aβ(1-42) Oligomers B]

A rat Aβ(1-42) oligomer A solution obtained according to example 2a isdiluted with 2.475 ml of water (0.05% SDS content, 0.1 mM rat Aβ(1-42))and incubated at 37° C. for 20 hours. Aliquots of this rat Aβ(1-42)oligomer B preparation can be frozen at −80° C. and stored for furtherstudies.

c) Alternative Preparation of Aβ(i42) Oligomers Having Molecular Weightsof 38 kDa and 48 kDa [(Aβ(1-42) Oligomers B]

An Aβ(1-42) oligomer A solution obtained according to example 2c isdiluted with 2.475 ml of water (0.125% lauric acid content, 0.1 mM Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42)oligomer B preparation can be frozen at −80° C. and stored for furtherstudies.

d) Alternative Preparation of A(1-42) Oligomers Having Molecular Weightsof 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]

An Aβ(1-42) oligomer A solution obtained according to example 2d isdiluted with 2.475 ml of water (0.125% oleic acid content, 0.1 mMAβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of thisAβ(1-42) oligomer B preparation can be frozen at −80° C. and stored forfurther studies.

e) Alternative Preparation of Aβ(1-42) Oligomers B Having MolecularWeights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]

An Aβ(1-42) oligomer A solution obtained according to example 2e isdiluted with 2.475 ml of water (0.125% laurylsarcosine content, 0.1 mMAβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of thisAβ(1-42) oligomer B preparation can be frozen at −80° C. and stored forfurther studies.

f) Preparation of SDS-Free Aβ(1-42) Oligomers B Having Molecular Weightsof 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]

10 μl of an Aβ(1-42) oligomer B preparation prepared according toexample 6b are admixed with 250 μl of an acetic acid/methanol/watermixture in a 4%/133%/163% ratio and incubated at 0° C. on ice for 30min. After centrifugation (10 000 g for 10 min), the supernatant isremoved and the precipitated protein residue taken up to 200 μl ofbuffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4). The preparationobtained in this way contains the dissolved Aβ(1-42) oligomers B inSDS-free from and can be stored at −20° C.

EXAMPLE 6

a) Dialysis and Concentration of Aβ(1-42) Oligomers Having MolecularWeights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]

An Aβ(1-42) oligomer B preparation prepared according to example 5a isadmixed with 30 ml of PBS buffer containing 0.1% Pluronic®F68 ( BASF)and concentrated to 3 ml in an Amicon Centriprep YM, 30 kD. Residueswhich may be present are removed by centrifugation (10 000 g for 5 min).The supernatant is removed. Aliquots of this Aβ(1-42) oligomer Bpreparation can be frozen at −80° C. and stored for further studies.

b) Preparation of a Concentrate of Aβ(1-42) Oligomers B Having MolecularWeights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]

72.6 ml of an Aβ(1-42) oligomer B preparation obtained according toexample 5a are concentrated to 2 ml via a 30 kD Centriprep YM Tube(Amicon). The concentrate is removed by centrifugation at 10 000 g for10 min. The supernatant is removed and ialyzed at 6° C. in a dialysistube against 1 l of buffer (5 mM sodium phosphate, 35 mM sodiumchloride, pH 7.4) for 16 h. The dialysate is removed by centrifugationat 10 000 g for 10 min. The supernatant is removed and can be stored at−80° C. for further studies.

EXAMPLE 7

Preparation of Biotin-Aβ(1-42) Stock Suspension

0.5 mg of biotin-β-amyloid(1-42) protein (short name: biotin-Aβ(1-42);peptide-synthetic material, lyophilisate, AnaSpec) are dissolved in 200μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorfvessel at 37° C. for 30 min. This is followed by evaporation to drynessin a vacuum concentrator (Speed Vac). The residue is taken up with 20.5μl of DMSO, producing a 5 mM biotin-A β(1-42) stock suspension which canbe stored at −20° C.

EXAMPLE 8

Preparation of Biotin-Aβ(1-42) Oligomers Having Molecular Weights of 17kDa and 22 kDa [biotin-Aβ(1-42) Oligomers A]

2 μl of the stock suspension of example 7 are admixed with 23 μl of PBSbuffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) and adjusted to anSDS content of 0.2% with 2.4 ,μl of 2% strength SDS solution. This isfollowed by 6 hours of incubation at 37° C. Insoluble components areremoved by centrifugation at 10 000 g for 5 min. The biotin-Aβ(1-42)oligomer A preparation obtained in this way can be stored at −20° C.

EXAMPLE 9

Preparation of Biotin-Aβ(1-42) Oligomers Hhaving Molecular Weights of 42kDa and 52 kDa [biotin-Aβ(1-42) Oligomers B]

A biotin-Aβ(1-42) oligomerA solution obtained according to example 8 isdiluted with 82 μl of water (0.05% SDS content, 0.1 mM Aβ) and incubatedat 37° C. for 16 hours. Insoluble components are removed bycentrifugation at 10 000 g for 5 min. The biotin Aβ(1-42) oligomer Bpreparation can be frozen at −20° C. and stored for further studies.(FIG. 3).

EXAMPLE 10

Preparation of Fluorescein-Aβ(1-42) Stock Suspension

0.5 mg of fluorescein-β-amyloid(1-42) protein (short name:fluorescein-Aβ(i-42); peptide-synthetic material, lyophilisate, AnaSpec)are dissolved in 200 μl of 1,1,1,3,3,3-hexafluoro-2-propanol andincubated in an Eppendorf vessel at 37° C. for 30 min. This is followedby evaporation to dryness in a vacuum concentrator (Speed Vac). Theresidue is taken up with 20.5 μl of DMSO, producing a 5 mMfluorescein-Aβ(1-42) stock suspension which can be stored at −20° C.

EXAMPLE 11

Preparation of Fluorescein-Aβ(1-42) Oligomers Having Molecular Weightsof 17 kDa and 22 kDa [Fluorescein-Aβ(1-42) Oligomer A]

2 μl of the stock suspension of example 10 are admixed with 23 μl of PBSbuffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) and adjusted to anSDS content of 0.2% with 2.4 μl of 2% strength SDS solution. This isfollowed by 6 hours of incubation at 37° C. Insoluble components areremoved by centrifugation at 10 000 9 for 5 min. Thefluorescein-Aβ(1-42) oligomer A preparation obtained in this way can bestored at −20° C.

EXAMPLE 12

Preparation of Fluorescein-Aβ(1-42) Oligomers Having Molecular Weightsof 42 kDa and 52 kDa [Fluorescein-Aβ(1-42) Oligomers B]

A fluorescein-Aβ(1-42) oligomer A solution obtained according to example11 is diluted with 82 μl of water (0.05% SDS content, 0.1 mM Aβ) andincubated at 37° C. for 16 hours. The fluorescein Aβ(1-42) oligomer Bpreparation can be frozen at −80° C. and stored for further studies.(FIG. 4).

EXAMPLE 13

Crosslinking of Aβ(1-42) Oligomers A of Example 2a [Aβ(1-42) OligomersA-CL]

10 μl of an Aβ(1-42) oligomer A solution prepared according to example2a are diluted to 100 μM Aβ(1-42) content with 7.5 μl of PBS, 0.2% SDS.To this solution 1 μl of freshly prepared 10 mM glutardialdehydesolution in water is added, followed by stirring at RT for 3 h. Theexcess glutardialdehyde is saturated by adding 1 μl of 100 mMethanolamine solution in water, pH 7.4, to the sample and stirring for 1h. The preparation obtained in this way contains crosslinked Aβ(1-42)oligomers A and is referred to as A3(1 -42) oligomer A-CL preparation.

EXAMPLE 14

a) Crosslinking of Aβ(1-42) Oligomers B of Example 5a [Aβ(1-42)Oligomers B-CL]

10 IL of an Aβ(1-42) oligomer B solution prepared according to example5a are admixed with 1 μl of freshly prepared 10 mM glutardialdehydesolution in water and stirred at RT for 3 h. The excess glutardialdehydeis saturated by adding 1 1μl of 100 mM ethanolamine solution in water,pH 7.4, to the sample and stirring for 1 h. The preparation obtained inthis way contains crosslinked Aβ(1-42) oligomers B and is referred to asAβ(1-42) oligomer B-CL preparation.

b) Alternative Procedure for Crosslinking Aβ(1-42) Oligomers [Aβ(1-42)Oligomers B-CL]

72.6 ml of an Aβ(1-42) oligomer B solution prepared according to example5a are admixed with 7.26 ml of freshly prepared 10 mM glutardialdehydesolution in water and stirred at RT for 2 h. The excess glutardialdehydeis saturated by adding 726 μl of buffer (20 mM sodium phosphate, 140 mMNaCl, 500 mM ethanolamine, pH 7.4) to the sample and stirring at RT for30 min. The reaction mixture is concentrated to 3 ml via a 15 ml 30 kDaCentriprep tube. The concentrate is removed by centrifugation at 10 000g for 10 min. The supernatant is removed and dialyzed at 6° C. in adialysis tube against 1 l of 5 mM sodium phosphate, 35 mM NaCl, pH 7.4for 16 h. The dialysate is subsequently removed by centrifugation at 10000 g for 10 min and the supernatant is removed and can be stored at−80° C. for further studies. The preparation obtained in this waycontains crosslinked Aβ(1-42) oligomers B and is referred to as Aβ(1-42)oligomer B-CL preparation.

EXAMPLE 15

a) Preparation of Truncated Aβ(20- 42) Oligomers, Starting From Aβ(1-42)Oligomers B, by Cleavage With Thermolysin:

1.59 ml of Aβ(1-42) oligomer B preparation prepared according to example6b are admixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 μlof a 1 mg/ml thermolysin solution (Roche) in water. The reaction mixtureis stirred at RT for 20 h. Then 80 μl of a 100 mM EDTA solution, pH 7.4,in water are added and the mixture is furthermore adjusted to an SDScontent of 0.01% with 400 1μl of a 1% strength SDS solution. Thereaction mixture is concentrated to approx. 1 ml via a 15 ml 30 kDaCentriprep tube. The concentrate is admixed with 9 ml of buffer (50 mMMES/NaOH, 0.02 % SDS, pH 7.4) and again concentrated to 1 ml. Theconcentrate is dialyzed at 6° C. against 1 l of buffer (5 mM sodiumphosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate isadjusted to an SDS content of 0.1% with a 2% strength SDS solution inwater. The sample is removed by centrifugation at 10 000 g for 10 minand the supernatant is removed.

The material thus obtained is analyzed further (SDS polyacrylamide gelelectrophoresis; cf. FIG. 11); the mass-spectrometric analysis of thetruncated oligomers produced reveals that the oligomer is composed oftruncated Aβ(20- 42) .

b) Preparation of Truncated Aβ(12- 42) Oligomers, Starting From Aβ(1-42)Oligomers B, by Cleavage With Endoproteinase GluC:

2 ml of an Aβ(1-42) oligomer B preparation prepared according to example6b are admixed with 38 ml buffer (5 mM sodium phosphate, 35 mM sodiumchloride, pH 7.4) and 150 μl of a 1 mglml GluC endoproteinase (Roche) inwater. The reaction mixture is stirred for 6 h at RT, and a further 150μl of a 1 mg/ml GluC endoproteinase (Roche) in water are subsequentlyadded. The reaction mixture is stirred at RT for another 16 h, followedby addition of 8 μl of a 5 M DIFP solution. The reaction mixture isconcentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. Theconcentrate is admixed with 9 ml of buffer (5 mM sodium phosphate, 35 mMsodium chloride, pH 7.4) and again concentrated to 1 ml. The concentrateis dialyzed at 6° C. against 1 l of buffer (5 mM sodium phosphate, 35 mMNaCl) in a dialysis tube for 16 h. The dialysate is adjusted to an SDScontent of 0.1% with a 1% strength SDS solution in water. The sample isremoved by centrifugation at 10 000 9 for 10 min and the supernatant isremoved.

The material thus obtained is analyzed further (SDS polyacrylamide gelelectrophoresis; cf. FIG. 11). Mass-spectrometric analysis of thetruncated oligomer produced reveal that the oligomer is composed oftruncated Aβ(12-42).

Characterization of Aβ(1 -42) Oligomers

EXAMPLE 16

SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

The molecular weight under denaturing conditions is characterized byanalyzing the preparations of examples 2a, 5a, 9, 12, 13 and 14a, all ofwhich contain oligomers A and B, under denaturing conditions accordingto standard conditions in a 4-20% strength Tris-glycine SDS PAGE.

The evaluation of said SDS PAGE (FIG. 1) reveals that starting proteinAβ(1-42) still present in the oligomer A preparation appears as a bandat about 4 kDa, while the oligomers Al and A2 of example 2a, denoted 1and 2, respectively, in FIG. 1, are visible at about 15 kDa (weakerband) and at about 20 kDa (main band).

In the oligomer B preparation comparatively little starting proteinAβ(1-42) can be detected (relatively weak band at about 4 kDa). Incontrast, the oligomers B1 and B2 of example 5a, denoted 3 and 4,respectively, in FIG. 1 appear at about 38 kDa and about 48 kDa (seearrows). Correspondingly higher molecular weights of about 42 kDa andabout 52 kDa arise for the biotin- and fluorescein-derivatized oligomersB of examples 9 and 12 (FIGS. 3, 4).

Analysis of the preparations containing the respective crosslinkingproducts A-CL and B-CL (examples 13 and 14a; FIG. 2) reveals that thetwo samples essentially maintain their degree of oligomerization (cf.lane A with A′and lane B with B′). The slightly different migrationbehavior compared to the oligomers A and B can be explained by aslightly altered SDS binding capacity and by modification of the aminogroups of the N terminus and, respectively, the lysine residue.

Analysis of the truncated Aβ(20-42) oligomers (of Example 15a) revealsthat the 38/48 kDa double band (FIG. 11, lane A) is converted to a 28/38kDa double band (Fig. 11; lane B) by proteolytic removal of theN-terminal peptide with themolysine. Similarly, analysis of thetruncated Aβ(12-42) oligomers (of Example 15b) reveals that the 38/48kDa double band (FIG. 11; lane A) is converted to a 33/40 kDa doubleband (Fig. 11; ring C) by proteolytic removal of the N-terminal peptidewith Glu-C endoprotease.

EXAMPLE 17

Gel Permeation Chromatography

In order to study the molecular weight behavior under nondenaturingconditions in more detail, a gel permeation chromatography (GPC) iscarried out by way of an FPLC process using a Superose 12 HR10/30column. The GPC is run at 4° C.

The column is equilibrated with 5 volumes of PBS buffer (flow rate 0.5ml/min, UV detection, 214 nm) and first calibrated using proteinstandards. Subsequently, the Aβ(1-42) oligomer B preparation of example5a (FIG. 5, bottom) and, for comparison, the same concentration offreshly weighed Aβ(1-42) lyophilisate dissolved in PBS are analyzed,after removing the insoluble components by centrifugation at 10 000 gfor 5 minutes (FIG. 5, top).

The evaluation reveals that the Aβ(1-42) oligomer B preparation has aprotein fraction characterized by a main peak in the molecular weightrange of around approximately 50 kDa. As under denaturing conditions inthe SDS PAGE, this protein fraction differs significantly from themonomeric Aβ(1-42) protein characterized by a main peak in the molecularweight range of around aproximately 16 kDa.

In order to study the molecular weight behavior of Aβ(1-42) oligomers Bof example 6b and of Aβ(1-42) oligomers B-CL of example 14a undernondenaturing conditions, a gel permeation chromatography (GPC) iscarried out by way of an FPLC process using a Superose 12 HR10/30 columnat room temperature. The column is equilibrated with 5 column volumes ofPBS buffer (flow rate 0.5 ml/min, UV detection, 214 nm) and firstcalibrated using protein standards. Subsequently, Aβ(1-42) oligomer B ofexample 6b is diluted to 1 mg/ml with PBS buffer and Aβ(1-42) oligomerB-CL of example 14a is diluted to 1 mg/ml with PBS buffer, and bothmixtures are analyzed (FIG. 12).

The evaluation reveals that the Aβ(1-42) oligomer B preparation withreduced SDS content has a protein fraction characterized by a main peakin the molecular weight range around approximately 100 kDa. Incomparison therewith, the Aβ(1-42) oligomer B-CL preparation withreduced SDS content has a protein fraction characterized by a main peakin the molecular weight range around approximately 60 kDa.

EXAMPLE 18

Native polyacrylamide gel electrophoresis (NATIVE PAGE) of Aβ(1-42)oligomers B of example 5a

The molecular weight under native conditions is characterized byanalyzing the oligomers B-containing preparations of example 5a undernondenaturing conditions in a 4-20% Tris-glycine gel.

The detergent present in the preparations is neutralized by the nonionicdetergent Triton X-100. To this end, 1 μl (4%Triton X-100) is pipettedto 10 μl of the preparation of the example 5a and incubated at roomtemperature for 5 min. Subsequently, 10 μl are admixed with the samevolume of native sample buffer (4 ml of 1M Tris, pH 6.8, 8 ml ofglycerol, 1 ml of bromophenol blue in 50 ml of H₂O) and theelectrophoresis (running buffer: 7.5 9 of Tris, 36 g of glycine to 2.5 1of H₂O).

The NATIVE PAGE evaluation reveals that the starting peptide Aβ(1-42)still present in the oligomer B preparation appears as a band at about28 kDa (lane A, denoted 1), while the main bands of the preparation ofexample 5a are visible at an apparent molecular weight of 64-90 kDa(lane A, denoted 2) (FIG. 6).

It is important that molecular weights can be assigned to the oligomersof the invention, in particular the oligomers B, also in nativemolecular weight analytical methods such as gel permeationchromatography (see example 17) or native gel electrophoresis (seeexample 18).

After complexing of the SDS, the oligomers B to which molecular weightsof about 38 and 48 kDa can be assigned in the SDS PAGE are detected inthe native gel electrophoresis as a band in the molecular weight rangeof about 64-90 kDa, based on selected standard proteins. This methodcannot be expected to provide an exact reading of the molecular weight,since the migration behavior of the oligomers is substantiallydetermined by their intrinsic charges, in addition to their size.However, the result leads to the conclusion that a defined oligomericspecies is present.

EXAMPLE 19

a) Stability of Aβ(1-42) Oligomers B at Various Protein Concentrationsin Physiological Buffers

The Aβ(1-42) oligomers B obtained according to example 6b are tested forstability in PBS buffer under the following conditions.

5 mg/ml are diluted with PBS in 2 x dilution steps down to 0.08 mg/ml.All solutions obtained are incubated at room temperature for 24 hours.This was followed by analyzing the band pattern in an SDS PAGE incomparison with a frozen control preparation. The band pattern isidentical in all samples.

b) Stability of Aβ(1-42) Oligomers B at Various Temperatures and AfterDifferent Periods of Time in Physiological Buffers

The A β(1-42) oligomers B obtained according to example 6b are dilutedto 0.5 mg/ml with PBS buffer and incubated at room temperature or at 37°C. for 24 h or 96 h.

Subsequently, the band pattern was analyzed in an SDS PAGE. The bandpattern is identical in all samples.

EXAMPLE 20

Proteolysis of Aβ(1-42) oligomers B by means of various proteases(trypsin, chymotrypsin, thermolysin, elastase, papain)

Aliquots of the Aps(1-42) oligomer B preparation obtained according toexample 6b are diluted to 0.5 mg/ml with buffer (20 mM sodium phosphate,140 mM NaCl, pH 7.4) and incubated with in each case 1/50 of the amountby weight of the protease solutions indicated in FIG. 9 under thefollowing conditions at 37° C. and pH 7.4 for 20 h. 1 pg of reactivemixture aliquots are then analyzed in an SDS-PAGE (FIG. 9).

The SDS PAGE reveals that, starting from the Aβ(1-42) oligomers Bpreparation, all proteases revert the double band at about 38/48 kDa toa kDa double band at about 32/28 kDa under the chosen limitedproteolysis conditions.

EXAMPLE 21

Stability of Aβ(1-42) Oligomers B In Rat Plasma

4 μl of the Aβ(1-42) oligomer B preparation prepared according toexample 6b are incubated with 76 μl of rat plasma at room temperaturefor 0 h, 1 h, 2 h, 4 h and 8 h. The incubations are stopped by freezingin dry ice.

Subsequently, all samples are analyzed in an SDS PAGE, after addition ofSDS sample buffer. This is followed by an evaluation of the stabilityvia staining of the Aβ(1-42) oligomers B in a Western blot. Theanti-Aβ(1-42) antibody 6E10 (Signet) was used for detection. The bandsare made visible by an anti-mouse IgG antibody coupled toalkaliphosphatase and addition of the substrate NBT/BCIP. Both molecularweight and intensity of the observed band pattern remain nearlyunchanged over a period of 2 h, 4 h and 8 h. This result suggests thatthe Aβ(1-42) oligomers B have high plasma stability. According to this,the biological half life in the plasma is in the range of about 8 hoursor longer.

EXAMPLE 22

Binding of Biotin-Aβ(1-42) Oligomers Having Molecular Weights of 17 kDaand 22 kDa [Biotin-Aβ(1-42) Oligomers A] and, Respectively, of 42 kDaand 52 kDa [Biotin-Aβ(1-42) Oligomers B] to the Surface of HumanNeuronal Cells.

Binding of human β-amyloid(1-42) protein and the two Aβ(1-42) oligomer Aand Aβ(1-42) oligomer B preparations to the human neuroblastoma cellline IMR-32 (ATCC Number: CCL-127) is studied by means of FACScan(Beckton Dickinson). A suspension of IMR-32 cells (1,5 ×10⁶ cells/0,1 mlPBS) is incubated with biotin-labeled human β-amyloid(1-42) protein(peptide-synthetic material, lyophilisate, AnaSpec) and the preparationscontaining the biotin-labeled oligomers A and B (examples 8 and 9,respectively) at 37° C. for 20 minutes. The cells are subsequentlywashed with buffer (PBS plus 1 % BSA) and incubated at room temperaturewith fluorescein coupled to streptavidin isothiocyanate (Sigma) for 20minutes. After a washing step with buffer, binding to the surface ofIMR-32 cells was analyzed by FACScan. The dashed line indicatesbackground fluorescence in the absence of the biotin-labeled components.The addition of the individual preparations resulted in a strongincrease of cell-associated fluorescence and is represented by the thickline. Binding of the oligomers A (FIG. 7B) and B (FIG. 7C) to the cellsurfaces is distinctly different from the binding of the monomericβ-amyloid(1-42) protein (FIG. 7A). The data indicate specific bindingsites for said oligomers on human cell surfaces.

EXAMPLE 23

Detection of Aβ(1-42) Oligomers B in Rat Brain Homogenates After IcvAdministration

10 nmol of an Aβ(1-42) oligomer B preparation obtained according toexample 6b are administered as icv-bolus to rats. The brains areprepared after 15 min and 120 min, respectively. Brains of untreatedcontrol animals are likewise prepared. For this purpose, in each case 1g of rat brain is admixed with 9 ml of disruption buffer A (200 ml of 5mM sodium phosphate, 35 mM NaCl, 300 mM sucrose, adjusted to pH 7.4, areadmixed with 4 tablets of Complete® protease inhibitor cocktail fromRoche) in a 50 ml Falcon tube and disrupted under ultrasound(UltraTurrax) on ice for 2 min. The solution is left standing for 20min, then shaken briefly and divided into 8 ×1 ml aliquots(=homogenate).

In order to carry out quantitative detection, first a series ofstandards of the Aβ(1-42) oligomer B preparation in PBS in theconcentration range of 1.58 ng/μl- 0.005 ng/ μl is prepared.

Furthermore, as a positive control, a homogenate from a control brain ofan untreated rat is also processed and a series of standards of theAβ(1-42) oligomer B preparation is prepared in this brain homogenate inthe same way. The homogenates are then introduced in an ultracentrifugeat 100 000 g for 1 h and the supernatants are used for the subsequentanalyses.

From the comparison of the values measured on both series of standards(PBS versus control brain-homogenate supernatant), the Aβ(1-42) oligomerB content can be determined quantitatively as follows, first in thepositive control and then, in comparison therewith, also in the brainsample of treated rats.

1) Detection by Dot Blot

1 μl drops of the series of standards samples and of the sample extractsfrom the treated animals are applied to nitrocellulose paper, andAβ(1-42) is detected using the antibody 6E10 (anti-Aβ(1-42); Signet).Staining is carried out using an alkaliphosphatase coupled to anti-mouseIgG and by adding the staining reagent NBT/BCIP.

While no Aβ(1-42) (0.01 nmol/g) is detectible in the brain extracts ofuntreated rats, about 0.4 nmol/g Aβ(1-42) can be detected in the ratssacrificed 15 min after treatment by comparing the staining intensitywith the corresponding concentrations of the positive control, andapprox. 0.2 nmol/g can still be detected by the same method in the ratssacrificed 120 min after treatment. This results in an averagebiological half life of about 105 min for the exogenously administeredAβ(1-42) oligomers B.

2) Detection by Western Blot

All dot blot-analyzed samples are likewise analyzed in a Western blot.The Western blot is likewise developed with mMAb 6E10 (anti-Aβ(1-42);Signet) and furthermore with an alkaliphosphatase coupled to anti-mouseIgG and by adding the staining reagent NBT/BCIP.

Anti-Aβ(1-42) reactive bands only occur in the 38/48 kDa region,corresponding to the apparent molecular weight of the Aβ(1-42) oligomersB, i.e. the oligomeric structure is still retained with in vivoadministration, even after 2 hours.

Furthermore, as in the dot blot method, the same concentrations can beestimated in the brains of the 15 min rats (0.4 nmol/g) and the 120 minrats (0.2 nmol/g) by comparing the staining intensities withcorrespondingly intensively stained bands of the Aβ(1-42) oligomers B inthe positive control.

EXAMPLE 24

Neurotoxic action of Aβ(1-42) oligomers having molecular weights of 38kDa and 48 kDa [Aβ(1-42) oligomers B] on murine cortical neurons

Murine cortical neurons are prepared and cultured as a mixed culturewith glia cells following the literature (Choi et al. (1987) J.Neurosci. 7, 357-368). The cortices of embryos on days 14-15 ofdevelopment are mechanically removed from the meninges and lower brainregions. The cells are separated from one another by incubation in a0.05% strength trypsin solution at 37° C. for 5-7 minutes andsubsequently pipetting said solution several times through a pasteurpipette with reduced opening. After determining the number of cells, 430000 cells are seeded in 0.5 ml of maintenance medium (minimum essentialmedium containing 0.8 mM glutamine, 18 mM glucose, 23 mM NaHCO₃ and 10%of horse serum) per 2 cm² on cell culture material coated withpoly-L-ornithine and laminine. Cultivation and later incubations of thecells are carried out in a humidified cell culture incubator at 37° C.,5% CO₂. After 3-5 days in culture, propagation of the glia cells isinterrupted by a 1-day incubation with a mixture of(+)-5-fluoro-2'-deoxyuridine/uridine (10μM each). After 14 days inculture, the toxic action of Aβ(1-42) oligomers B is studied. For thispurpose, the cells are incubated in brain cell buffer (120 mM NaCl, 5.4mM KCI, 1.8 mM CaCl₂, 15 mM glucose, 25 mM HEPES, pH 7.2) for 15minutes. Control cells 1 are incubated with 300 μM L-glutamate in braincell buffer for the same time. The Aβ-oligomer stock solution is dilutedwith serum-free medium (minimum essential medium containing 0.8 mMglutamine, 20 mM glucose, 26 mM NaHCO₃) to various final concentrationsand incubated for 24 h. The L-glutamate-treated control cells 1 areincubated in parallel in serum-free medium. Another group of cells(control cells 2) is treated only with brain cell buffer and serum-freemedium. The cell culture supernatants are removed after 24 h, theremaining cells are destroyed by incubation in distilled water for 20minutes and the activity of the enzyme lactate dehydrogenase (LDH) isdetermined enzymatically in both solutions. For evaluation, the ratio ofthe LDH activity of the cell culture supernatants to the sum of the LDHactivities of supernatant and remaining cells is determined and theaverage of the quadruple determinations is formed. 3 experiments arecarried out, with each experimental condition being present inquadruplicate (n =12). The average for the glutamate-treated cells(control cells 1) is set to 100% neuronal death, the average of thecells which have been treated neither with L-glutamate nor with Aβoligomers (control cells 2) is set to 0% neuronal death and the valuesof the cells treated with Aβ oligomers are converted accordingly. Theneurotoxicity average of all determinations at the concentrations usedin each case indicates a distinct toxic action of the Aβ(1-42) oligomershaving molecular weights of 38 kDa and 48 kDa [Aβ(1-42) oligomer B], cf.FIG. 8.

EXAMPLE 25

Production of Antibodies

The cocktails used for immunization contain in all cases adjuvant(Biogenes) with essentially the following components:

-   -   95% Paraffin oil    -   2.4% Tween 40    -   0.1% Cholesterol    -   0.1% Lipopolysaccharide

The adjuvant is mixed with a solution of the antigen in a 2:1 ratiountil a stable emulsion is obtained. The emulsion is injected and formsa depot from which said antigen is released steadily.

a) Production of Polyclonal Antisera by Immunization of Rabbits WithTruncated Aβ(20- 42) Oligomers B of Example 15a.

2 rabbits are immunized with unconjugated Aβ(20-42) oligomer Bpreparation of example 15a according to a standard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum (a1).

2 mg of Aβ(20-42) oligomer B preparation of example 15a are coupled toLPH with glutardialdehyde under standard conditions and 2 more rabbitsare immunized with this conjugated Aβ(20-42) oligomer B according to astandard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(a2).

b) Production of Polyclonal Antisera by Immunization of Rabbits WithTruncated Aβ(12- 42) Oligomers B of Example 15b.

2 rabbits are immunized with unconjugated Aβ(12-42) oligomer Bpreparation of example 15b according to a standard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(bl).

2 mg of Aβ(12- 42) oligomer B preparation of example 15b are coupled toLPH with glutardialdehyde under standard conditions and 2 more rabbitsare immunized with this conjugated Aβ(12- 42) oligomer B according to astandard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(b2).

c) Production of Polyclonal Antisera by Immunization of Rabbits WithAβ(1-42) Oligomer Preparation B-CL of Example 14a.

2 rabbits are immunized with unconjugated Aβ(1-42) oligomers B-CLpreparation of example 14a according to a standard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(c1).

d) Production of Polyclonal Antisera by Immunization of Rabbits With Aβ(1-42) Oligomer Preparation B of Example 6b.

2 rabbits are immunized with unconjugated Aβ(1-42) oligomers Bpreparation of example 6b according to a standard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(d1).

2 mg of Aβ(1-42) oligomer B preparation of example 6b are coupled toLPH, with glutardialdehyde under standard conditions and 2 more rabbitsare immunized with this conjugated Aβ(1-42) oligomer B preparationaccording to a standard protocol:

Day 1 Primary immunization and taking of preimmune serum

Day 7 First boost

Day 14 Second boost

Day 28 Third boost and bleeding

Day 35 Taking blood

The antisera are obtained from the rabbit's blood by letting the latterstand at room temperature and subsequent centrifugation at roomtemperature. The serum obtained in this way is referred to as serum(d2).

EXAMPLE 26

Characterization of polyclonal antisera from the immunizations ofexample 25 regarding their cross reaction with various Aβ(1-42) forms.

In order to characterize the polyclonal antisera, serial dilution in therange of 100 pmol/μl-0.01 pmol/μl of the various Aβ(1-42) forms wereprepared in PBS. In each case 1 μl of the sample was applied to anitrocellulose membrane: detection was carried out using the appropriaterabbit sera of example 25. A detection using mMAb 6E10 (Signet) wascarried out for comparison. Alkaliphosphatase coupled to anti-rabbit IgG(for comparison: alkaliphosphatase coupled to anti-mouse IgG) was usedfor staining together with addition of the staining reagent NBT/BCIP.FIG. 10 indicates the dot blots obtained in this manner, which can benumerically evaluated as follows: Serum Antigen 6E10 Serum (a1) Serum(a2) Serum (d1) (c1) Oligomer 0.1 0.5 10 0.1 0.5 Monomer 0.1 10 100 0.11 Fibril 1 100 >100 0.5 1 APP 0.01 >1 1 0.1 0.1 Aβ(1-40)* 0.1 100 n.d.0.5 10*not shown in FIG. 10

The numbers in the table indicate the minimum amount of antigen visible[in pmol; in each case based on the monomer, except for APP](detectionlimit).

Comparison of the immunological reactions with rabbit serum (al)obtained by immunization with unconjugated Aβ(20- 42) oligomer ofexample 15a clearly indicates that the antibodies directed against theseoligomers cross-react only weakly with the other forms such as fibrils,APP and monomer. In contrast, a markedly stronger cross reaction withthe Aβ(1-42) oligomer B (see row 1) and, moreover, a cross reaction withthe stabilized CL antigen are observed. This data indicates a distinctlydifferent structure of the oligomer form present here, in comparisonwith APP, monomer and the fibril structure. The antibodies bind tooligomer-specific structures.

The immunological reactions with rabbit serum (a2) obtained byimmunization with conjugated Aβ(20- 42) oligomer of example 1 5alikewise show that antibodies directed against these oligomerscross-react only weakly with the other forms such as fibrils andmonomer. In contrast, a weak cross reaction with the Aβ(1-42) oligomer B(see row 1) and, moreover, a cross reaction with the stabilized CLantigen and with APP are observed. This data indicates a likewisedistinctly-different structure of the oligomer form present here, incomparison with monomer and the fibril structure.

In comparison therewith, the immunological reactions with rabbit serum(d1) obtained by immunization with A β(1-42) oligomer of example 6b, andalso with rabbit serum (c1) obtained by immunization with Aβ(1-42)oligomer B-CL of example 14a indicate that the antibodies directedagainst these oligomers cross-react with the other forms such asfibrils, APP and monomer comparably as strongly as with the Aβ(1-42)oligomers B (see row 1). These antibodies exhibit an immune reactioncomparable to that of mMAb 6E10 (Signet) and do not bind tooligomer-specific structures. Correspondingly, mice were immunized withAβ(1-42) oligomers of example 6b and monoclonal antibodies establishedin a manner known per se. Here, 2 out of 10 hybridomas secretedmonoclonal antibodies whose binding profiles are similar to those of theantiserum (a1), in particular with respect to the reactivites assayedabove.

EXAMPLE 27

Preparation of Aββ(1-42) Fibrils

100 μl of 2 mg/ml A β(1-42) solution in 0.1% NH₄0H are diluted with 300μl of buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) to 0.5 mg/mland adjusted to pH 7.4 with 0.1 M HCl (100 μM Aβ(1-42)). The sample isincubated at 37° C. for 24 h and then removed by centrifugation at 10000 g for 10 min. The protein residue obtained is resuspended with 400μl of buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4). The Aβ(1-42)fibril preparation obtained in this way can be stored at −20° C. andused for further studies.

EXAMPLE 28

Effects of Aβ(1-42) Oligomers B-CL (of Example 14b) on HippocampalSlices

Transversal hippocampal slices of 400 μM in thickness were adapted in asubmersion slice chamber at 34° C. under perfusion by gassed Ringer'ssolution (NaCl 124 mM, KCl 4.9 mM, MgSO₄ 1.3 mM, CaCl₂ 2.5 mM, KH₂PO₄1.2 mM, NaHCO₃ 25.6 mM, glucose 10 mM). Subsequently, the Schaffercollateral was stimulated with the aid of a monopolar stimulatingelectrode and the excitatory postsynaptic potential (fEPSP) in thestratum radiatum was recorded. Test pulses were given with 30% of thestimulus level that generates a maximum fEPSP. Long-term potentiationwas induced by applying 100 pulses three times every 10 minutes, withsingle pulses having a width of 200 μs (strong tetanus). The resultingpotentiation of the fEPSPs was recorded for at least 240 minutes. Therinsing-in of the oligomer B-CL of example 14b (500 nM) started 20minutes before the first tetanus and was stopped 10 minutes after thelast tetanus. The rise (slope) of the fEPSPs was determined and plottedas a function of time.

Figure FIG. 14 indicates that the washing-in of Aβ(1-42) oligomerssuppresses long-term potentiation in hippocampus, especially in themaintenance phase. Accordingly, the A β(1-42) oligomers BCL influencethe storage of information by nerve cells (cellular memory).

EXAMPLE 29

Binding of Aβ(1-42) oligomers B (of example 5b) to rat primaryhippocampal neurons The binding of rat Aβ(1-42) oligomers B of example5b to rat primary hippocampal neurons was studied. The hippocampalneurons were cultured in neurobasal medium with B27 supplement onpoly-L-lysine-coated cover slips and used on day 14 of the culture. Theoligomers were bound to the cell membrane of the neurons by adding 200nM (total monomeric Aβ concentration) oligomers to fresh culture mediumand incubating at 37° C. for 15 minutes. After removing theAβ-containing medium, two washing steps with medium were carried out andthe cells were then fixed in 3.7% formaldehyde. After further washing ofthe cells, unspecific binding sites were blocked with 10% normal donkeyserum in PBS buffer at room temperature for 90 min. 6E10 (from mouse)was applied as the first antibody in a 1:2000 dilution at roomtemperature for 2 h. The cells were washed again and incubated with thesecond antibody (from donkey) which is directed against mouse and whichis coupled to the fluorescent dye Cy3, at room temperature for 2 h.After the cells had been washed again, the cover slips containing theneurons were fixed to a slide with embedding medium. The hippocampalneurons with bound Aβ oligomers were depicted in a fluorescentmicroscope. The control used was a mixture in which the first antibody6E10 had been omitted. This control thus exhibits unspecificfluorescence not based on Aβ.

As FIG. 15 a shows, the oligomers bind to the cell surface of theneurons in a dotted manner. In contrast, the control without the firstantibody 6E10 exhibits only low unspecific fluorescence, due to bindingof the second antibody to the neurons (FIG. 15 b).

1. An oligomer of the amyloid β(1-42) protein. selected from the groupconsisting of (A1) an oligomer having an apparent molecular weight ofabout 15 kDa in SDS gel electrophoresis; (A2) an oligomer having anapparent molecular weight of about 20 kDa in SDS gel electrophoresis;(B1) an oligomer having an apparent molecularweight of about 38 kDa inSDS gel electrophoresis: and (B2) an oligomer having an apparentmolecular weight of about 48 kDa in SDS gel electrophoresis, or aderivative thereof, or a composition comprising (i) a mixture of theoligomer (A1) or the derivative thereof and (ii) the oligomer (A2) orthe derivative thereof, or a composition comprising (i) a mixture of theoligomer (B1) or the derivative thereof and (ii) the 6ligomer (B2) orthe derivative thereof.
 2. (canceled)
 3. (canceled)
 4. (canceled) 5.(canceled)
 6. (canceled)
 7. The oligomer or composition as claimed inclaim 1, characterized in that the derivatives have a detectable label.8. The oligomer or composition as claimed in claim 7, characterized inthat the label is a fluorescent, luminescent, calorimetric, radioactiveor magnetic label or is a label having affinity to complementary bindingpartners.
 9. The oligomer or composition as claimed in a claim 1,characterized in that the oligomer or the derivative is crosslinked. 10.The oligomer or composition as claimed in claim 1 characterized in thatthe derivative is obtainable by proteolytic cleavage of the oligomer.11. The oligomer or composition as claimed in claim 10, characterized inthat the derivative is an oligomer of an A β(x-42) fragment, where x is8 to 24, preferably from 10 to 22, and in particular from 12 to
 20. 12.The oligomer or composition as claimed in claim 11, characterized inthat the derivative is an oligomer of the Aβ(12-42) fragment or of theAβ(20-42) fragment.
 13. A method for preparing oligomers of aderivatized or nonderivatized amyloid β(1-42) protein, characterized inthat monomeric amyloid β(1-42) protein or a derivative thereof isexposed to a detergent and, optionally, at least one oligomer orderivative thereof is obtained in a manner known per se.
 14. The methodas claimed in claim 13, characterized in that, subsequent to theexposure of the monomeric amvloid β(1-42) protein or a derivativethereof to the detergent, the detergent action is reduced and incubationis continued.
 15. The method as claimed in claim 13, characterized inthat the detergent is ionic.
 16. The method as claimed in claim 15,characterized in that the detergent is sodium dodecyl sulfate.
 17. Themethod as claimed in claim 13 characterized in that the time of exposureto the detergent is from about 1 to 20 hours.
 18. The method as claimedin claim 17, characterized in that the temperature of exposure is fromabout 20 to 50° C.
 19. The method as claimed in claim 1, characterizedin that the time of continued incubation is from 10 to 30 hours.
 20. Themethod as claimed in claim 19, characterized in that the temperature ofexposure is from about 20 to 50° C.
 21. The method as claimed in claim13, characterized in that the amyloid β(1-42) protein is unfolded atleast partially beforehand.
 22. The method as claimed in claim 21,characterized in that for this purpose the protein is exposed to ahydrogen bond-breaking agent, preferably HFIP.
 23. The method as claimedin claim 13, characterized in that the oligomer is exposed to acrosslinker.
 24. The method as claimed in claim 13, characterized inthat the oligomer is exposed to a protease.
 25. The use of an oligomer,of a derivative thereof or of a composition as claimed in claim 1 in adiagnostic in vitro detection method.
 26. The use of an oligomer, of aderivative thereof or of a composition as claimed in claim 1 in adiagnostic in vivo detection method.
 27. The use of an oligomer, of aderivative thereof or of a composition as claimed in claim 1 forgenerating oligomer-specific antibodies.
 28. A method for characterizinga substance or a substance mixture, which comprises i) providing saidsubstance or said substance mixture; ii) exposing an oligomer, aderivative thereof or a composition as claimed in claim 1 to saidsubstance or said substance mixture; and iii) determining, whether saidsubstance or particular portions of said substance mixture bind to saidoligomer, said derivative thereof or to at least one oligomer orderivative thereof present in said composition.
 29. A substance or aportion of a substance mixture, characterized in that said substance orsaid portion is identifiable by a method as claimed in claim 28 as aligand binding to the oligomer, the derivative thereof or to at leastone oligomer or derivative thereof present in the composition.
 30. Amethod for the treatment of amyloid βassociated, in particulardementing, disorders, which comprises administering an effective amountof a substance or of a portion of a substance mixture as claimed inclaim 29 to a subiet in need thereof.
 31. A method for the diagnosis ofamyloid β-associated, in particular dementing, disorders, whichcomprises administering an effective amount of a substance or of aportion of a substance mixture as claimed in claim 29 to a subiet inneed thereof.
 32. A method for producing antibodies, which comprises i)immunizing a host with at least one oligomer, derivative thereof orcomposition as claimed in claim 1 and ii) obtaining anantibody-containing host serum produced as a response to saidimmunization.
 33. The method as claimed in claim 32, characterized inthat at least one antibody of the serum is selected, which recognizesthe oligomer or derivative thereof used as immunogen or at least oneoligomer or derivative thereof present in the composition used asimmunogen.
 34. A method for preparing antibodies with specificity for anoligomer or derivative thereof as claimed in at claim 1, which comprisesi) providing an antigen comprising said oligomer or derivative thereof;ii) exposing an antibody repertoire to said antigen; and iii) selectingfrom said repertoire an antibody which specifically binds to saidoligomer or derivative thereof.
 35. The method as claimed in claim 34,wherein the antibody repertoire is exposed to the antigen in vivo byimmunizing an animal with said antigen.
 36. The method as claimed inclaim 34, wherein furthermore a number of hybridomas from lymphocytes ofthe animal are established and a hybridoma secreting an antibody whichspecifically binds to the oligomer or derivative thereof is selected.37. The method as claimed in claim 34, wherein the animal is selectedfrom among mice, rats, chickens, camelids, cynomolus monkeys, rabbitsand goats.
 38. The method as claimed in claim 37, wherein the animal isa knockout mouse with APP deficiency.
 39. The method as claimed in claim37, wherein the animal is a transgenic mouse having human immunoglobulingenes, which generates human antibodies after an antigenic stimulus. 40.The method as claimed in claim 37, wherein the animal is a mouse withsevere combined immunodeficiency (SCID), which has been reconstitutedwith human peripheral mononuclear blood cells or lymphoid cells orprecursors thereof.
 41. The method as claimed in claim 37, wherein theanimal is a mouse which has been treated with lethal total bodyirradiation, then protected against radiation with bone marrow cellsfrom a mouse with severe combined immunodeficiency (SCID) and into whichsubsequently functional human lymphocytes or precursors thereof havebeen transplanted.
 42. The method as claimed in claim 34, wherein theantibody repertoire is exposed to the antigen in vitro by screening arecombinant antibody library with said antigen.
 43. The method asclaimed in claim 42, wherein the recombinant antibody library isexpressed on the surface of a bacteriophage.
 44. The method as claimedin claim 42, wherein the recombinant antibody library is expressed onthe surface of yeast cells.
 45. The method as claimed in claim 42,wherein the recombinant antibody library is expressed on the surface ofbacterial cells.
 46. The method as claimed in claim 42, wherein therecombinant antibody library is expressed as RNA-protein fusions. 47.The method as claimed in claim 42, wherein the recombinant antibodylibrary is an scFv library or an Fab library.
 48. The method as claimedin claim 42, wherein the recombinant antibody library is a single domainlibrary.
 49. The method as claimed in claim 34, wherein the antibodyrepertoire is exposed to the antigen by immunizing an animal in vivowith said antigen and a recombinant antibody library prepared from thelymphoid cells of said animal is then screened with said antigen. 50.The method as claimed in claim 34, wherein the antibody repertoire isexposed to the antigen by immunizing an animal in vivo with said antigenand a recombinant antibody library prepared from the lymphoid cells ofsaid animal is then subjected to affinity maturation in vitro.
 51. Themethod as claimed in claim 34, wherein the antibody repertoire isexposed to the antigen by immunizing an animal with said antigen andindividual cells secreting the antigen-binding antibody are thenselected and cDNAs for the variable regions of the heavy and lightchains are obtained from the single cells.
 52. An antibody, obtainableby a method as claimed in claim
 32. 53. A protein, which bindsspecifically to an oligomer or derivative thereof as claimed in anclaim
 1. 54. The protein as claimed in claim 53, which binds theoligomer or derivative thereof with an affinity in the range ofK_(D)=10⁻⁶−10⁻¹² M.
 55. The protein as claimed in claim 54, which bindsthe oligomer or derivative thereof with an affinity greater thanK^(d)=10⁻⁸ M.
 56. The protein as claimed in claim 54, which binds theoligomer or derivative thereof with an affinity greater than K^(d)=10⁻⁹M.
 57. The protein as claimed in claim 54, which binds the oligomer orderivative thereof with an affinity greater than K^(d)=10⁻¹⁰ M.
 58. Theprotein as claimed in claim 54, which binds the oligomer or derivativethereof with an affinity greater than K^(d)=10⁻¹¹ M.
 59. The protein asclaimed in a x claims 53 to 58, which binds monomeric Aβ(1-42) proteinand/or monomeric Aβ(1-40) protein with an affinity of less thanK^(d)=10⁻⁸ M.
 60. The protein as claimed in ano claim 53, wherein theaffinity of said protein to the oligomer or derivative thereof is atleast 10 times greater than to monomeric Aβ(1-42) protein and/ormonomeric Aβ(1-40) protein.
 61. The protein as claimed in anyof claim53, wherein the affinity of said protein to the oligomer or derivativethereof is at least 100 times greater than to monomeric Aβ(1-42) proteinand/or monomeric Aβ(140) protein.
 62. The protein as claimed in an claim53, wherein the affinity of said protein to the oligomer or derivativethereof is at least 1000 times greater than to monomeric Aβ(1-42)protein and/or monomeric A β(1-40) protein.
 63. The protein as claimedin any of claim 53, which binds the oligomer or derivative thereof witha rate constant k_(off) of 0.1 s⁻¹ or lower, as determined by means ofsurface plasmon resonance.
 64. The protein as claimed in any of claim53, which inhibits the activity of the oligomer or derivative thereofwith an inhibition constant IC₅₀ of 1×10⁻⁵ or lower.
 65. The protein asclaimed in an claim 53, which is an antibody or an antigen-bindingmoiety thereof.
 66. The protein as claimed in claim 53, which is asubstantially human antibody or an antigen-binding moiety thereof. 67.The protein as claimed in claim 53, which is a chimeric antibody or anantigen-binding moiety thereof.
 68. The protein as claimed in claim 53,which is a CDR graft antibody or an antigen-binding moiety thereof. 69.The protein as claimed in claim 53, which is a molecule derived from theT-cell receptor or a receptor domain derived from the T-cell receptor ora fusion protein of said receptor domain with an Fc moiety of animmunoglobulin.
 70. A pharmaceutical agent, comprising a protein asclaimed in claim 52 or 53 and, optionally, a pharmaceutically suitablecarrier.
 71. A method for the treatment of amyloid β-associated, inparticular dementina. disorders, which comprises administering aneffective amount of a protein as claimed in claim 52 or 53 to a subiectin need thereof.
 72. A method for the treatment of amvloid 1-associated,in particular dementing. disorders, which comprises administering aneffective amount of a protein as claimed in claim 52 or 53 to a subiectin need thereof.
 73. A vaccine, comprising at least one oligomer or atleast one derivative thereof or a composition as claimed in claim
 1. 74.A method for the treatment of amyloid β-associated, in particulardementing, disorders, which comprises administering an effective amountof a vaccine as claimed in claim 73 to a sublet in need thereof.